Human Oncostatin M Antibodies and Methods of Use

ABSTRACT

Antibodies and compositions capable of neutralizing oncostatin M biological functions are useful in treating diseases and disorders associated with oncostatin M, such as osteoarthritis and idiopathic pulmonary fibrosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/392,683, filed 13 Oct. 2010, the entire contents of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a human antibody capable ofneutralizing the biological activity produced by oncostatin M binding tomembrane receptors on human cells, and uses.

BACKGROUND OF THE INVENTION

Oncostatin M (OSM) is a 28 kDa multifunctional member of the IL-6 familyof cytokines secreted by monocytes, macrophages, neutrophils andactivated T-lymphocytes (Tanaka & Miyajima, Rev Physiol BiochemPharmacol 149: 39-53, 2003). Proteolytic cleavage near thecarboxy-terminus of the secreted OSM yields the fully active form ofOSM, 209 amino acids length having two N-linked glycosylation sites. OSMbelongs to the IL-6 family of cytokines that includes (IL-6, IL-11,leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neutotrophicfactor (CNTF) and cardiotrophin-like cytokine (CLC)) which share acommon receptor subunit, gp130 protein. In humans, OSM signals throughreceptor heterodimers consisting of gp130 and the LIFRα subunit or gp130and the OSMRβ subunit. In contrast to the other cytokines of the IL-6family, OSM binds gp130 directly and in the absence of any additionalmembrane-bound co-receptor (Gearing et al., Science 255: 1434-1437,1992). Following OSM binding to gp130, OSMRβ or LIFRα are recruited toform a high-affinity signaling complex (Mosley et al., J Biol Chem 271:32635-32643, 1996). Activation of either receptor results in signalingvia the JAK/STAT pathway (Auguste et al., J Biol Chem 272: 15760-15764,1997).

OSM is produced primarily by cells of immune system origin and, becauseof the widespread distribution of its signaling receptors, it has beenassociated with a variety of biological activities, including cellgrowth regulation, neural development and regulation of extracellularmatrix composition.

As its name implies, oncostatin M is associated with oncogenicprocesses. However, OSM is also involved in early events in inflammatoryand hypertrophic pathways leading to deleterious conditions, such aspulmonary fibrosis. Thus, there is a need to provide human antibodiesspecific for human OSM capable of blocking receptor signaling (gp130signaling) events which signal blocking antibodies can exert aclinically useful cytotoxic, cytostatic, or immunomodulatory effects ongp130 expressing cells.

SUMMARY OF THE INVENTION

The present invention provides OSM binding, monoclonal antibodiescapable of blocking activities associated with one or more bioactivitiesassociated with OSM and OSM binding receptor interaction on cells,tissues, or organs in a host subject. Amino acid sequences of exemplaryOSM binding monoclonal antibodies are provided which can be encoded bynucleic acids for expression in a host cell. One or more of the OSMmonoclonal antibodies of the invention define an epitope on the surfaceof OSM which, when engaged by an antibody of the invention, is preventedfrom interaction with the receptor components of the signaling complex,gp130 and LIFRα or gp130 and OSMRβ, thereby preventing ligand ligationdriven signaling and downstream biological activity.

One aspect of the invention is an isolated antibody reactive with humanOSM protein having the antigen binding ability of a monoclonal antibodycomprising an antigen binding domain comprising amino acid sequences asset forth in SEQ ID NOs: 13-18 alone or at specified positions ofFR1-CDR1-FR2-CDR2-FR3 as set forth in SEQ ID NOs: 1-3, a CDR3 asrepresented by SEQ ID NO: 27-29 and 47; or an antigen binding domaincomprising amino acid sequences as set forth in SEQ ID NOs: 23-26 aloneor at specified positions as set forth in SEQ ID NOs: 5-8 and variantsthereof, and a CDR3 as represented by SEQ ID NO: 19-22. In a specificembodiment, the human OSM binding antibody comprises a variable domainselected from SEQ ID NO: 49-55.

In another embodiment of the invention, the monoclonal antibody bindingdomains used as full length IgG structures, have constant domainsderived from human IgG constant domains or specific variants thereof andare used as therapeutic molecules in a pharmaceutical preparation toprevent binding of OSM to cells displaying OSM receptor components. Inanother embodiment, the binding domains are configured as antibodyfragments for use as a therapeutic molecule capable of prevent bindingof OSM to cells displaying OSM receptor components. In one aspect of theinvention, there is provided a pharmaceutically acceptable formulation,delivery system, or kit or a method of treating oncostatin M-relatedconditions comprising one or more of the OSM binding domains of theinvention such as but not limited to 13-28 and 30-46 and variants asprovided by SEQ ID NO: 29 and 47.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1 shows the germline gene sequences used to build the Fab librariesdisplayed on pIX coat protein, wherein each of the HV domains consist ofa variegated FR1-CDR1-FR2-CDR2-FR3 according to SEQ ID NO: 1-3(1=IGHV1-69, 2=IGHV3-23, and 3=IGHV5-51) followed by a variable length,varigated H-CDR3 region, and a J-region (FR4, SEQ ID NO: 4); and each ofthe LV domains consist of a variegated FR1-CDR1-FR2-CDR2-FR3 accordingto SEQ ID NO: 5-8 (5=IGKV1-39 (O12), 6=IGKV3-11 (L6), 7-IGKV3-20 (A27),and 8=IGKV4-1 (B3)) followed by a CDR3 according to SEQ ID NO: 9 whichis followed by a J-region (FR4, SEQ ID NO: 10).

FIG. 2A is a graph of the dose-response of the CHO cell-derivedrecombinant human and cynomolgous monkey oncostatin M in suppressingproliferation of A375-S2 cells as measured by BrdU incorporation andnormalized to the control in the presence of vehicle only.

FIG. 2B is a graph showing the ability of the antibody M71, comprised ofthe L180 (SEQ ID NO: 53) and H17 (SEQ ID NO: 54) variable domains torelieve OSM suppression of A375-S2 proliferation where OSM was presentat a concentration of 2 ng/ml.

FIG. 3 is a column graph showing the effect of M64, M71, M55, and M69 at20 μg/ml in increasing ³⁵SO₄-uptake, a measure of increased proteoglycansynthesis, above the level observed in the absence of antibody and wherethe non-specific isotype antibody was a control in co-cultures of humanchondrocytes in alginate beads and human macrophages capable ofsecreting OSM.

FIG. 4A is a graph in showing the dose response of human OSM stimulatedpSTAT3 in NHLF cells where the EC50 was found to be approximately 1ng/ml.

FIG. 4B is a graph showing the ability of the antibody M71 to neutralizethe pSTAT3 signal in the presence of 2 ng/ml OSM.

FIGS. 5A and B are scatter plots showing the amount of IP-10 (A) andMCP-1 (B) detected in the serum of individual mice after challenge withOSM and with or without pretreatment with the indicated concentration ofM71 antibody.

FIGS. 6A and B are graphs showing the serum concentration over time incynomolgous monkeys after intravenous (A) or subcutaneous (B)administration of 3 mg/Kg M71 or an Fc variant of M71.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though fully set forth.

ABBREVIATIONS

BSA=bovine serum albumin; CDR=complementarity determining region;Cyno=Cynomolgus monkey (Macaca fascicularis); DN=diabetic nephropathy;ECD=extracellular domain; FR=framework; H=heavy chain; IPF=interstitialpulmonary arthritis; L=light chain; Ig=immunoglobulin; Mab=monoclonalantibody; OSM=oncostatin M; OA=osteoarthritis; PBS=phosphate bufferedsaline; RA=rheumatoid arthritis; VL=Variable light chain; VH=Variableheavy chain,

DEFINITIONS

As used herein, an “antibody” includes whole antibodies and any antigenbinding fragment or a single chain thereof. Thus, the antibody includesany protein or peptide containing molecule that comprises at least aportion of an immunoglobulin molecule, such as but not limited to, atleast one complementarity determining region (CDR) of a heavy or lightchain or a ligand binding portion thereof, a heavy chain or light chainvariable region, a heavy chain or light chain constant region, aframework (FR) region, or any portion thereof, or at least one portionof a binding protein, which can be incorporated into an antibody of thepresent invention. The term “antibody” is further intended to encompassantibodies, digestion fragments, specified portions and variantsthereof, including antibody mimetics or comprising portions ofantibodies that mimic the structure and/or function of an antibody or aspecified fragment or portion thereof, including single chain and singledomain antibodies and fragments thereof. Functional fragments includeantigen-binding fragments to a preselected target. Examples of bindingfragments encompassed within the term “antigen binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH, domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH,domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (scFv); see e.g., Bird et al. (I 988) Science 242:423-426, and Hustonet al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such singlechain antibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies. Conversely, libraries of scFv constructs can beused to screen for antigen binding capability and then, usingconventional techniques, spliced to other DNA encoding human germlinegene sequences. One example of such a library is the “HuCAL: HumanCombinatorial Antibody Library” (Knappik, A. et al. J Mol Biol (2000)296(1):57-86).

The term “CDR” refers to the complementarity determining region orhypervariable region amino acid residues of an antibody that participatein or are responsible for antigen-binding. The hypervariable regions orCDRs of the human IgG subtype of antibody comprise amino acid residuesfrom residues 24-34 (L-CDR1), 50-56 (L-CDR2) and 89-97 (L-CDR3) in thelight chain variable domain and 31-35 (H-CDR1), 50-65 (H-CDR2) and95-102 (H-CDR3) in the heavy chain variable domain as described by Kabatet al. (1991 Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.)and/or those residues from a hypervariable loop (i.e., residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) or the current H2 Chothia definition of 52-57,and 96-101 (H3) in the heavy chain variable domain as described by(Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). Chothia and Leskrefer to structurally conserved HVs as “canonical structures.” Frameworkor FR1-4 residues are those variable domain residues other than andbracketing the hypervariable regions. The numbering system of Chothiaand Lesk takes into account differences in the number of residues in aloop by showing the expansion at specified residues denoted by the smallletter notations, e.g., 30a, 30b, 30c, etc. More recently, a universalnumbering system has been developed and widely adopted, internationalImMunoGeneTics information System® (IMGT) (LaFranc, et al. 2005. NuclAcids Res. 33:D593-D597).

Herein, the CDRs are referred to in terms of both the amino acidsequence and the location within the light or heavy chain by sequentialnumbering. As the “location” of the CDRs within the structure of theimmunoglobulin variable domain is conserved between species and presentin structures called loops, by using numbering systems that alignvariable domain sequences according to structural features, CDR andframework residues and are readily identified. This information is usedin grafting and replacement of CDR residues from immunoglobulins of onespecies into an acceptor framework from, typically, a human antibody.

The term “maturation” is applied to directed changes in an antibodyvariable region for the purpose of altering the properties of thepolypeptide. As is known in the art and described herein, a large numberof positions in the V-region sequences that can impact recognition ofantigen. In nature, antibodies achieve high affinity and specificity bythe progressive process of somatic mutation. This process can beimitated in vitro to permit parallel selection and targeted variationwhile maintaining the sequence integrity of each antibody chain suchthat they reflect the species, in the present case, a human, antibody,while enhancing affinity or a biophysical parameter such as solubilityor resistance to oxidation. The process of making directed changes or“maturation” is typically performed at the level of the coding sequenceand can be achieved by creating sublibraries for selection of theenhanced property.

As used herein “OSM” refers to an oncostatin M polypeptide orpolynucleotide comprising a coding sequence encoding the OSMpolypeptide. Human OSM is the product of the human osm gene (Gene 5008).

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules, such as amino acids or sugar side chainsand usually have specific three-dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

As used herein, K_(D) refers to the dissociation constant, specifically,the antibody K_(D) for a predetermined antigen, and is a measure ofaffinity of the antibody for a specific target. High affinity antibodieshave a K_(D) of 10⁻⁸ M or less, more preferably 10 M or less and evenmore preferably 10⁻¹⁰ M or less, for a predetermined antigen. Thereciprocal of K_(D) is K_(A), the association constant. The term“k_(dis)” or “k₂,” or “k_(d)” as used herein, is intended to refer tothe dissociation rate of a particular antibody-antigen interaction. The“K_(D)” is the ratio of the rate of dissociation (k₂), also called the“off-rate (k_(off))” to the rate of association rate (k₁) or “on-rate(k_(on)).” Thus, K_(D) equals k₂/k₁ or k_(off)/k_(on) and is expressedas a molar concentration (M). It follows that the smaller the K_(D), thestronger the binding. Thus, a K_(D) of 10⁻⁶ M (or 1 microM) indicatesweak binding compared to 10⁻⁹ M (or 1 nM).

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope. Theterm also includes “recombinant antibody” and “recombinant monoclonalantibody” as all antibodies are prepared, expressed, created or isolatedby recombinant means, such as (a) antibodies isolated from an animal ora hybridoma prepared by the fusion of antibody secreting animal cellsand an fusion partner, (b) antibodies isolated from a host celltransformed to express the antibody, e.g., from a transfectoma, (c)antibodies isolated from a recombinant, combinatorial human or otherspecies antibody library, and (d) antibodies prepared, expressed,created or isolated by any other means that involve splicing ofimmunoglobulin gene sequences to other DNA sequences. An “isolatedantibody,” as used herein, is intended to refer to an antibody which issubstantially free of other antibodies having different antigenicspecificities. An isolated antibody that specifically binds to anepitope, isoform or variant of human OSM may, however, havecross-reactivity to other related antigens, e.g., from other species(e.g., OSM species homologs). Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals. In oneembodiment of the invention, a combination of “isolated” monoclonalantibodies having different specificities are combined in a well definedcomposition.

As used herein, “specific binding,” “immunospecific binding” and “bindsimmunospecifically” refers to antibody binding to a predeterminedantigen. Typically, the antibody binds with a dissociation constant(K_(D)) of 10⁻⁷ M or less, and binds to the predetermined antigen with aK_(D) that is at least twofold less than its K_(D) for binding to anon-specific antigen (e.g., BSA, casein, or any other specifiedpolypeptide) other than the predetermined antigen. The phrases “anantibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen.” As used herein “highlyspecific” binding means that the relative K_(D) of the antibody for thespecific target epitope is at least 10-fold less than the K_(D) forbinding that antibody to other ligands.

As used herein, “type” refers to the antibody class (e.g., IgA, IgE, IgMor IgG) that is encoded by heavy chain constant region genes. Someantibody classes further encompass subclasses or “isotypes” which arealso encoded by the heavy chain constant regions (e.g., IgG1, IgG2, IgG3and IgG4). Antibodies may befurther decorated by oligosaccharides linkedto the protein at specific residues within the constant region domainswhich further enhance biological functions of the antibody. For example,in human antibody isotypes IgG1, IgG3 and to a lesser extent, IgG2,display effector functions as do murine IgG2a antibodies.

By “effector” functions or “effector positive” is meant that theantibody comprises domains distinct from the antigen specific bindingdomains capable of interacting with receptors or other blood componentssuch as complement, leading to, for example, the recruitment ofmacrophages and events leading to destruction of cells bound by theantigen binding domains of the antibody. Antibodies have severaleffector functions mediated by binding of effector molecules. Forexample, binding of the C1 component of complement to antibodiesactivates the complement system. Activation of complement is importantin the opsonisation and lysis of cell pathogens. The activation ofcomplement stimulates the inflammatory response and may also be involvedin autoimmune hypersensitivity. Further, antibodies bind to cells viathe Fc region, with a Fc receptor site on the antibody Fc region bindingto a Fc receptor (FcR) on a cell. There are a number of Fc receptorswhich are specific for different classes of antibody, including IgG(gamma receptors), IgE (eta receptors), IgA (alpha receptors) and IgM(mu receptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), release of inflammatory mediators, placental transfer and controlof immunoglobulin production.

The term “polypeptide” means a molecule that comprises amino acidresidues linked by a peptide bond to form a polypeptide. Smallpolypeptides of less than 50 amino acids may be referred to as“peptides.” Polypeptides may also be referred as “proteins.”

Overview

The present invention provides isolated Mabs capable of binding andneutralizing the biological activity of OSM proteins and cleavageproducts. In particular, the OSM binding mAbs of the invention are ableto block OSM binding to gp130 or prevent the recruitment of LIFRα orOSMRb by OSM bound gp130. In either case, the OSM mAb of the inventionis capable of blocking OSM driven gp130 receptor signaling.

Phosphorylation of STAT3 has been reported to lead to over-production ofcollagen by fibroblasts in a variety of pathological contexts (Lim etal. Oncogene 23(39): 5416-25, 2006; Huang et al. J Cell Biochem 81(1):102-13, 2001). These properties demonstrate the potential therapeuticvalue of these antibodies for RA, OA and for fibrotic indications suchas idiopathic pulmonary fibrosis (IPF) and diabetic nephropathy (DN).

The human OSM gene product, OSM (NCBI Accession No. NP_(—)065391) is apre-pro-polypeptide 252 amino acids in length (SEQ ID NO: 11), having asignal peptide 25 amino acids in length and a proteolytic cleavage sitebetween residues 234 and 235. It is a secreted protein having fivecysteine residues forming two internal disulfides between residues 31 to152 and 74 to 192 (Kallestad J C, et al. J Biol. Chem. 1991 May 15; 266(14):8940-5). There are two potential N-linked glycosylation sites atresidues 100 and 217, and when produced in eukaryotic cells, the proteinis glycosylated. The human OSM has a free sulfhydryl at residue 105.

The sequence of cyno OSM protein was not available in the public domainalthough an automated computationally generated record for an 1867 bpmRNA (NCBI No. XM_(—)001110148) derived from an annotated genomicsequence (NW_(—)001095169) existed. To obtain the cyno OSM sequence, RNAwas isolated from cyno PBMC and the gene was then amplified from thiscDNA by RT-PCR and sequenced. The predicted translation of the clonedsequence (SEQ ID NO: 12) was found to be 99.6% identical to thepredicted Macaca mullata (Rhesus) sequence, 92% identical to the humanOSM protein sequence, and 41% identical to the mouse OSM proteinsequence as disclosed in applicants copending application (U.S. Ser. No.12/648,430).

Therefore, the present invention is directed toward the identificationof human derived OSM-binding Mabs capable of inhibiting downstreambiologic activity resulting from OSM bound gp130 signaling and whereinthe Mabs exhibit the ability to;

restore proliferation of cells in the presence of OSM, inhibitOSM-driven chondrocyte degradation of intra-articular (joint) matrix intissue explants,efficiently neutralize OSM-dependent STAT3 phosphorylation in human lungfibroblasts and prevent OSM induced cytokine release.

1. Composition of an Antibody of the Invention

An OSM-neutralizing antibody of the invention is an antibody thatinhibits, blocks, or interferes with at least one OSM activity or OSMreceptor binding, in vitro, in situ and/or in vivo and does not promote,stimulate, induce, or agonize OSM activity or ligand binding nor doesantibody binding mimic the downstream effects of OSM-driven ligation ofOSM receptors, in particular gp130 interaction with OSM, such as signaltransduction in a host cell. A suitable OSM-neutralizing antibody,specified portion, or variant can also, optionally, affect at least oneOSM activity or function, such as but not limited to; RNA, DNA orprotein synthesis; protein release; cell activation, proliferation ordifferentiation; antibody secretion; OSM receptor signaling; OSMcleavage; OSM binding, OSM or gp130 induction, synthesis or secretion.

The present invention is based upon the discovery of anti-human OSMmonoclonal antibodies capable of inhibiting gp130 signaling after OSMbinding or LIFR recruitment by OSM. Antibody binding domains in the formof a Fab library displayed on filamentous phage particles linked to thepIX coat protein (see WO29085462A1 and further described hereinbelow)were selected for the ability to bind OSM. A competition assay usinggp130 was used to distinguish those Fabs that, when bound to OSM,prevented OSM from binding gp130. Alternatively, Fabs were able toprevent LIFR recruitment to gp130 binding when bound to OSM. Acell-based (A375, human melanoma cell) assay was used to identifyseveral candidate antibodies capable of inhibiting gp130-mediated pSTAT3activation of OSM expressing host cells.

The OSM-binding antibodies described herein recognize at least twodistinct regions on the active form of human OSM protein, indicating theadditional discovery of multiple sites on OSM suitable for the targetingof antibodies or other compounds with similar function blockingcapabilities. Thus, expression and purification of the antibody bindingdomains provided herein as amino acid sequences further provides a toolwhich can provide the means for selection of novel molecules exhibitingOSM-neutralizing activity.

In one embodiment, the anti-human OSM antibody, has a binding regioncomprising a light chain variable (VL) or heavy chain variable (VH)region comprising the amino acid sequence as shown in SEQ ID NO: 49-55,and which antibody or binding portion thereof immunospecifically bindsOSM. In another embodiment of the invention, comprising a heavy chaincomprising SEQ ID NO: 54 or 55 an antigen binding portion thereof, bindsto OSM protein and, additionally, has the specified functionalproperties of antibodies of the invention, such as:

-   -   1. binds human OSM with a K_(D) of less than 100 pM    -   2. binds cyno OSM with a K_(D) of less than 500 pM    -   3. Is capable of restoring proliferation of A375-S2 cells in the        presence of 2 ng/ml human OSM by 90% of the level in the absence        of OSM,    -   4. Is capable of restoring proliferation of A375-S2 cells in the        presence of 2 ng/ml cyno OSM by 90% of the level in the absence        of OSM,    -   5 inhibits OSM-driven chondrocyte degradation of intra-articular        (joint) matrix in tissue explants,    -   6. efficiently neutralizes OSM-dependent STAT3 phosphorylation        in normal human lung fibroblasts (NHLF), or    -   7. blocks cytokine release after systemic challenge with OSM in        mice.

In another aspect of the invention, the structural features of theantibodies exhibiting some or all of the above referenced biologicalactivity as described herein and, in particular, the Mabs designated asM55 and M71 binding domains, are used to create structurally relatedhuman anti-OSM antibodies that retain at least one functional propertyof the antibodies of the invention, such as binding to OSM. Morespecifically, one or more CDR regions of M55 and M71 (such as specifiedresidues of SEQ ID NO: 1 and 8) can be combined recombinantly with knownhuman framework regions and CDRs such as SEQ ID NO: 13-28, 30-46 tocreate additional, recombinantly-engineered, human anti-OSM antibodiesof the invention.

In one embodiment, the antibodies of the invention have the sequences,including FR1, 2, and/or 3; of IGVH1-69 (SEQ ID NO: 1) or of IGVH5-51(SEQ ID NO: 3), wherein one or more residues from CDRs selected from thegroup consisting of SEQ ID NO: 13-22 are present in the CDR position ofSEQ ID NO: 1 or 3, while still retaining the ability of the antibody tobind OSM (e.g., conservative substitutions). Accordingly, in anotherembodiment, the engineered antibody may be composed of one or more CDRsthat are, for example, 90%, 95%, 98% or 99.5% identical to the CDRslisted in SEQ ID NOs: 13-22 or the variants of L-CDR3 as given by SEQ IDNO: 29 or 47.

In addition to simply binding OSM, engineered antibodies, such as thosedescribed above, may be selected for their retention of other functionalproperties of antibodies of the invention, such as the ability toinhibit binding of OSM protein or a cleavage product thereof to GP130positive cells to, which binding would result in suppression ofproliferation of the GP130 positive cells in vivo.

Human monoclonal antibodies of the invention can be tested for bindingto OSM by, for example, standard ELISA.

2. Generation of OSM-Neutralizing Antibodies

A OSM-neutralizing antibody exhibiting the desired bioactivity spectrumas exemplified herein by M5, M6, M9, M10, M42, M45, M53, M54, M55, M62,M63, M65, M66, M67, M68, M69, M71, and M83 comprising the heavy chainand light chain sequences as specified which comprise the libraryframeworks SEQ ID NOS: 1, 3, 8, and having CDRs of SEQ ID NOS: 13-28,30-46 can be generated by a variety of techniques.

In another embodiment, the epitope bound by the antibodies of theinvention, comprising as few as five to all of residues 51-227 of SEQ IDNO: 11 or a nucleic acid coding sequence therefore, can be used toimmunize a subject in order to produce the antibodies of the inventiondirectly in the host for the purpose of treating, preventing, orameliorating disease or symptoms of disease associated with theproduction of OSM.

In one embodiment and as exemplified herein, the human antibody isselected from a phage library, where that phage comprises humanimmunoglobulin genes and the library expresses human antibody bindingdomains as, for example, single chain antibodies (scFv), as Fabs, orsome other construct exhibiting paired or unpaired antibody variableregions (Vaughan et lo al. Nature Biotechnology 14:309-314 (1996):Sheets et al. PITAS (USA) 95:6157-6162 (1998)); Hoogenboom and Winter,J. Mol. Biol., 227:381 (1991); Marks et al. J. Mol. Biol., 222:581(1991)). Human monoclonal antibodies of the invention can also beprepared using phage display methods for screening libraries of humanimmunoglobulin genes. Such phage display methods for isolating humanantibodies are established in the art. See for example: U.S. Pat. Nos.5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos.5,427,908 and 5, 580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404;6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Phage clones are selected by and identified through a multi-stepprocedure known as biopanning. Biopanning is carried out by incubatingphage displaying protein ligand variants (a phage display library) witha target, removing unbound phage by a washing technique, andspecifically eluting the bound phage. The eluted phage are optionallyamplified before being taken through additional cycles of binding andoptional amplification that enriches the pool of specific sequences infavor of those phage clones bearing antibody fragments that display thebest binding to the target. After several rounds, individual phageclones are characterized, and the sequences of the peptides displayed bythe clones are determined by sequencing the corresponding DNA of thephage virion.

Fab Phage-pIX Library

In a specific embodiment of the phage display technology, a syntheticFab library displayed on the pIX phage coat protein, described in Shi etal. J Mol Biol 397:385-396, 2010; WO29085462A1 and U.S. Ser. No.12/546,850 and to be further detailed herein, is used to select binderfrom a repertoire of human IgG sequences derived from human germlinegenes. Libraries were constructed on four VL and three VH domainsencoded by known IGV and IGJ germline sequences selected based on thefrequency which the sequences have been observed to be present in humanantibodies isolated from natural sources. The VH, IMGT nomenclature,selected are IGHV1-69 (SEQ ID NO: 1), IGHV3-23 (SEQ ID NO: 2), orIGHV5-51 (SEQ ID NO: 3). The diversity in the VH design produces heavychains with variable length sequence in the CDR3 region with limiteddiversity positions in the H-CDR1 and H-CDR2 which remain at a constantlength. Framework four (H-FR4) is held constant among all members of thelibrary (SEQ ID NO: 4).

VH169, IGHV1-69*01  Length = 98, CDR1 = 31-35, CDR2 = 50-66(SEQ ID NO: 1) QVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYX ₁ ISWVRQA PGQGLEWMGX₂  IX ₃ X ₄ X ₅ X ₆ GTANY AQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCARWhere, in the 169 library X₁ is A or G, X₂ is G or W, X₃ may be I or S,X₄ may be P or A, X₅ may be I or Y and X₆ may be F or N.

VH323, IGHV3-23*01  Length = 98, CDR1 = 31-35, CDR2 = 50-66(SEQ ID NO: 2) EVQLLESGGG LVQPGGSLRL SCAASGFTFS X ₁ YX ₂ MX ₃WVRQAPGKGLEWVSX₄  IX ₅ X ₆ X ₇ GX ₈ STYY ADSVKGRFTI SRDNSKNTLYLQMNSLRAED TAVYYCAKWhere, in the 323 library, X₁ may be S, D, N, or T; X₂ may be A, G, orW; X₃ may be S or H; X₄ may by V, A, N or G; X₅ may be S, N, K or W; X₆may be Y, S, G, or Q; X₇ may be S or D; and X₈ may be S or G.

VH551, IGHV5-51*03  Length = 98, CDR1 = 31-35, CDR2 = 50-66(SEQ ID NO: 3) EVQLVQSGAE VKKPGESLKI SCKGSGYSFT X ₁ YWIX ₂WVRQMPGKGLEWMGX ₃ IX ₄ PX ₅ DSX ₆ TRY SPSFQGQVTI SADKSISTAYLQWSSLKASD TAMYYCARWhere in the 551 library, X₁ may be S, N, or T; X₂ may be S or G; X₃ maybe I or R; X₄ may by D or Y; X₅ may be G or S; X₆ may be D or Y.

A FR4 or JH region having (11 residues), WGQGTLVTVSS (SEQ ID NO: 4), hasbeen joined to the above sequences to form a complete heavy chainvariable region.

The H-CDR1 and H-CDR2 positions that were targeted for diversificationwere determined by 1) diversity in germline genes; and 2) frequencyfound in contact with antigen in antibody-antigen complexes of knownstructure (Almagro J Mol. Recognit. 17:132-143, 2004). The amino aciddiversity at the selected positions was determined by 1) usage ingermline; 2) amino acids that are most frequently observed in humanrearranged V genes; 3) amino acids predicted to be result from singlebase somatic mutations; and 4) biochemical and biophysical properties ofamino acids that contribute to antigen recognition.

The library incorporates diversity in the CDR3 of the VH(H3) mimickingthe repertoire of human antibodies (Shi et al. 2010 supra) as shownbelow (FORMULA I) where the final length is between 7 and 14 residues.Among the CDR3 of over 5000 human variable regions, amino acids glycine(G) and alanine (A) are frequently used in all positions. In addition,aspartic acid (D) is frequently used in position 95 and tyrosine (Y) isfrequently encoded in the positions preceding the canonical region ofthe J segment. Amino acids phenylalanine (F), aspartic acid (D) andtyrosine (Y) predominate at positions 99-101 used in IgGs at thesepositions. Since these positions often serve as structural support toH-CDR3 and are less accessible to antigen and/or to surface of IgG,amino acids phenylalanine plus leucine (50/50 ratio) at position 99,aspartic acid at position 100 and tyrosine at position 101 were fixed.Thus, the sequence of Formula I is inserted between SEQ ID NOS: 1, 2, or3 and SEQ ID NO: 4 to create a complete VH.

-(D)-(N)n(N+O)m(F)DY-  (I)

Where:

(D)=Asp (D) and Gly (G) rich position.(N)n=Ala (A) and Gly (G) rich position, n=3-7.(O)m=Ala (A), Gly (G) and Y (Tyr) rich in, m=1-4.(F)=The Phe (F) dominant position.

Various versions of the library encompass pairings with fixed ordiversified light chains derived also from the human germlinerepertoire. In the present invention, the four light-chain libraryVL_(kappa) genes (Kawasaki et al. 2001. Eur J Immunol 31: 1017-1028 andafter Schaeble & Zachau, 1993 Biol Chem Hoppe Seyler 374: 1001-1022) areA27 (IGKV3-20*01), B3 (IGKV4-1*01), L6 (IGKV3-11*01), and 012(IGKV1-39*01) where the gene name in parentheses are the presumedcorresponding IMGT gene. The Fabs are displayed on pIX via expression ofa dicistronic vector wherein the VH-CH1 domain is fused to the coatprotein sequence and the VL-CLkappa or VL-CLlambda is expressed as afree polypeptide which self-associates with the VH-CH1. The CDR regionsare underlined.

Light Chain Variable Library Based on Vkappa (Vk) Germline Genes

012; IGKV1-39*01, IGKV1D-39*01  Length = 88; CDR1 = 24-34, CDR2 = 50-56(SEQ ID NO: 5) DIQMTQSPSS LSASVGDRVT ITCRASQSIS X ₁ X ₂ X ₃ NWYQQKPGKAPKLLIYX ₄  ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYC L6, IGKV3-11*01 Length = 88, CDR1 = 24-34, CDR2 = 50-56 (SEQ ID NO: 6)EIVLTQSPAT LSLSPGERAT LSCRASQSV X ₁ X ₂ X ₃ LAWYQQKP GQAPRLLIYX₄ ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYC A27, IGKV3-20*01,  Length =89, CDR1 = 24-35, CDR2 = 51-57 (SEQ ID NO: 7)EIVLTQSPGT LSLSPGERAT LSCRASQSVX ₁ X ₂ X ₃ X ₄ LAWYQQK PGQAPRLLIY X ₅ASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCB3, DPK24, VKIVKlobeck; IGKV4-1*01  Length = 94, CDR1 = 24-40, CDR2 =56-62 (SEQ ID NO: 8) DIVMTQSPDS LAVSLGERAT INCKSSQSVL X ₁ SSNNX₂NX ₃ LAWYQQKPGQPP KLLIYX ₄ ASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYC

The diversity at the specified positions for each variable regionscaffold are summarized in Table 1 below where the amino acidssingle-letter code is used and is present in the alternative at thespecified positions as shown in FIG. 1.

TABLE 1 Kabat O12 L6 A27 B3 CDR Position (SEQ ID NO: 5) (SEQ ID NO: 6)(SEQ ID NO: 7) (SEQ ID NO: 8) 1 30 X₁ SRNAD X₁ SRNAD X₁ SRNTD L 30a — —X₂ SNR X₁ YSHFA 30e — — — 30f X₂ KTNE 31 X₂ SNKDG X₂ NSKD X₃ SNRADH N 32X₃ YHNDWF X₃ YWDFH X₄ YFHQSEK X₃ YFHNWDAS SAV SAN 2 50 X₄ FYTNKA X₄ADKGY X₅ ADGS X₄ WSRDYA DG FTN

The VL CDR3 in all of the libraries has seven residues wherein the firsttwo residues are glutamine (Gln, Q) and the residue corresponding toKabat residue 95 is proline (Pro, P). For L-CDR3 the sequencecorresponds to QQX₁X₂X₃X₄PX₅T (SEQ ID NO: 9), where varigation are as inthe table below and at the residue positions are according to Kabat.

TABLE 2 Kabat Res- CDR3 idue Position O12 L6 A27 B3 X₁ 91 SAYHPD RYSGFYSHA YSHA X₂ 92 FIYHNDKGRE RHNSL YNDSHIFKG YNDSHIFKG X₃ 93 STHNDRG NDKRSNTDGHR SNTDGHR X₄ 94 TYLVFSRGPI WA TYLVFAS TYLVFAS X₅ 96 LWRFYIN WYFLIRWYFLIR WYFLIR

As the variable sequence varies in length from gene to gene, diversityin a particular residue location within a hypervariable loop or CDR canbe described as follows using the residue numbering as defined inAl-Lazikani B, Lesk A M, Chothia C, 1997 (Standard conformations for thecanonical structures of immunoglobulins. J Mol Biol 273: 927-948). Inthis system, the changes in length of the hypervariable loops areaccommodated by the designation of subpositions a, b, c, etc. for agiven residue.

A framework 4 (FR4) segment such as JK4, FGQGTKVEIK (SEQ ID NO: 10) wasused to form a complete human light chain variable region.

Fab affinity for diverse protein targets from 0.2 to 20 nM has beendemonstrated in initial selections.

Methods for an integrated maturation process for improving bindingparameters consisting of reshuffling VL or VH diversity or,alternatively, directed or limited VL modification are accomplishedusing the vectors and primers designed and used for the libraries asdescribed in the referenced publication, as taught herein, and combinedwith what it known in the art.

Alternative Sources of OSM-Binding Immunoglobulin Domains

OSM binding antibodies with the characteristics of the human Mabsdisclosed herein may be made or binding fragments sourced fromimmunoglobulin domains formed by a number of methods, including thestandard somatic cell hybridization technique (hybridoma method) ofKohler and Milstein (1975) Nature 256:495. In the hybridoma method, amouse or other appropriate host animal, such as a hamster or macaquemonkey, is immunized as described herein to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization. Alternatively, lymphocytesmay be immunized in vitro. Lymphocytes then are fused with myeloma cellsusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,pp. 59-103 (Academic Press, 1986)).

An OSM-neutralizing antibody can also be optionally generated byimmunization of a transgenic animal (e.g., mouse, rat, hamster,non-human primate, and the like) capable of producing a repertoire ofhuman antibodies, as described herein and/or as known in the art. Cellsthat produce a human anti-OSM antibody can be isolated from such animalsand immortalized using suitable methods, such as the methods describedherein. Alternatively, the antibody coding sequences may be cloned,introduced into a suitable vector, and used to transfect a host cell forexpression and isolation of the antibody by methods taught herein andthose known in the art.

The use of transgenic mice carrying human immunoglobulin (Ig) loci intheir germline configuration provide for the isolation of high affinityfully human monoclonal antibodies directed against a variety of targetsincluding human self antigens for which the normal human immune systemis tolerant (Lonberg, N. et al., U.S. Pat. No. 5,569,825, U.S. Pat. No.6,300,129 and 1994, Nature 368:856-9; Green, L. et al., 1994, NatureGenet. 7:13-21; Green, L. & Jakobovits, 1998, Exp. Med. 188:483-95;Lonberg, N. and Huszar, D., 1995, Int. Rev. Immunol. 13:65-93;Kucherlapati, et al. U.S. Pat. No. 6,713,610; Bruggemann, M. et al.,1991, Eur. J. Immunol. 21:1323-1326; Fishwild, D. et al., 1996, Nat.Biotechnol. 14:845-851; Mendez, M. et al., 1997, Nat. Genet. 15:146-156;Green, L., 1999, J. Immunol. Methods 231:11-23; Yang, X. et al., 1999,Cancer Res. 59:1236-1243; Brüggemann, M. and Taussig, M J., Curr. Opin.Biotechnol. 8:455-458, 1997; Tomizuka et al. WO02043478). The endogenousimmunoglobulin loci in such mice can be disrupted or deleted toeliminate the capacity of the animal to produce antibodies encoded byendogenous genes. In addition, companies, such as Abgenix, Inc.(Freemont, Calif.) and Medarex (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology as described above.

Preparation of Polypeptides for Use as Target Ligands in Panningstrategies and as immunogenic antigens can be performed using anysuitable technique, such as recombinant protein production. The targetligand or fragment thereof in the form of purified protein, or proteinmixtures including whole cells or cell or tissue extracts, or, in thecase of an immunization, the antigen can be formed de novo in theanimal's body from nucleic acids encoding said antigen or a portionthereof.

The isolated nucleic acids of the present invention can be made using(a) recombinant methods, (b) synthetic techniques, (c) purificationtechniques, or combinations thereof, as well-known in the art. DNAencoding the monoclonal antibodies is readily isolated and sequencedusing methods known in the art (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of human antibody regions). Where a hybridoma is produced,such cells can serve as a source of such DNA. Alternatively, usingdisplay techniques wherein the coding sequence and the translationproduct are linked, such as phage or ribosomal display libraries, theselection of the binder and the nucleic acid is simplified. After phageselection, the antibody coding regions from the phage can be isolatedand used to generate whole antibodies, including human antibodies, orany other desired antigen binding fragment, and expressed in any desiredhost, including mammalian cells, insect cells, plant cells, yeast, andbacteria.

Human Antibodies

The invention further provides human immunoglobulins (or antibodies)which bind human OSM. These antibodies can also be characterized asengineered or adapted. The immunoglobulins have variable region(s)substantially from a human germline immunoglobulin and include directedvariations in residues known to participate in antigen recognition, e.g.the CDRs of Kabat or the hypervariable loops as structurally defined.The constant region(s), if present, are also substantially from a humanimmunoglobulin. The human antibodies exhibit K_(D) for OSM of at leastabout 10⁻⁶ M (1 microM), about 10⁻⁷ M (100 nM), 10⁻⁹ M (1 nM), or less.To affect a change in affinity, e.g., improve affinity or reduce K_(D),of the human antibody for OSM, substitutions in either the CDR residuesor other residues may be made.

The source for production of human antibody which binds to OSM ispreferably the sequences provide herein as the variable regions,frameworks and/or CDRs, noted as SEQ ID NO: 13-55 identified as capableof binding human OSM and cross-reacting with cynomolgous monkey OSMusing a repertoire of human derived Fab displayed on filamentous phageparticles.

The substitution of any of the CDRs into any human variable domainframework is most likely to result in retention of their correct spatialorientation if the human variable domain framework adopts the same orsimilar conformation to the parent variable framework from which theCDRs originated. The heavy and light chain variable framework regions tobe paired in the final Mab can be derived from the same or differenthuman antibody sequences. The human antibody sequences can be thesequences of naturally occurring human antibodies, be derived from humangermline immunoglobulin sequences, or can be consensus sequences ofseveral human antibody and/or germline sequences.

Suitable human antibody sequences are identified by computer comparisonsof the amino acid sequences of the mouse variable regions with thesequences of known human antibodies. The comparison is performedseparately for heavy and light chains but the principles are similar foreach.

With regard to the empirical method, it has been found to beparticularly convenient to create a library of variant sequences thatcan be screened for the desired activity, binding affinity orspecificity. One format for creation of such a library of variants is aphage display vector. Alternatively, variants can be generated usingalternate and known methods for randomizing or variegating a nucleicacid sequence encoding the targeted residues within the variable domain.

Another method of determining whether further substitutions arerequired, and the selection of amino acid residues for substitution, canbe accomplished using computer modeling. Computer hardware and softwarefor producing three-dimensional images of immunoglobulin molecules arewidely available. In general, molecular models are produced startingfrom solved structures for immunoglobulin chains or domains thereof. Thechains to be modeled are compared for amino acid sequence similaritywith chains or domains of solved three dimensional structures, and thechains or domains showing the greatest sequence similarity is/areselected as starting points for construction of the molecular model. Thesolved starting structures are modified to allow for differences betweenthe actual amino acids in the immunoglobulin chains or domains beingmodeled, and those in the starting structure. The modified structuresare then assembled into a composite immunoglobulin. Finally, the modelis refined by energy minimization and by verifying that all atoms arewithin appropriate distances from one another and that bond lengths andangles are within chemically acceptable limits.

Because of the degeneracy of the code, a variety of nucleic acidsequences will encode each immunoglobulin amino acid sequence. Thedesired nucleic acid sequences can be produced by de nova solid-phaseDNA synthesis or by PCR mutagenesis of an earlier prepared variant ofthe desired polynucleotide. All nucleic acids encoding the antibodiesdescribed in this application are expressly included in the invention.

The variable segments of human antibodies produced as described hereinare typically linked to at least a portion of a human immunoglobulinconstant region. The antibody will contain both light chain and heavychain constant regions. The heavy chain constant region usually includesCH1, hinge, CH2, CH3, and, sometimes, CH4 domains.

The human antibodies may comprise any type of constant domains from anyclass of antibody, including IgM, IgG, IgD, IgA and IgE, and anysubclass (isotype), including IgG1, IgG2, IgG3 and IgG4. When it isdesired that the humanized antibody exhibit cytotoxic activity, theconstant domain is usually a complement-fixing constant domain and theclass is typically IgG₁. When such cytotoxic activity is not desirable,the constant domain may be of the IgG₂ class. The humanized antibody maycomprise sequences from more than one class or isotype.

Nucleic acids encoding humanized light and heavy chain variable regions,optionally linked to constant regions, are inserted into expressionvectors. The light and heavy chains can be cloned in the same ordifferent expression vectors. The DNA segments encoding immunoglobulinchains are operably linked to control sequences in the expressionvector(s) that ensure the expression of immunoglobulin polypeptides.Such control sequences include a signal sequence, a promoter, anenhancer, and a transcription termination sequence (see Queen et al.,Proc. Natl. Acad. Sci. USA 86, 10029 (1989); WO 90/07861; Co et al., J.Immunol. 148, 1149 (1992), which are incorporated herein by reference intheir entirety for all purposes).

3. Methods of Using an Anti-OSM Antibody

As described in detail below, the present invention demonstrates thatisolated monoclonal antibodies having the variable domains of M5, M6,M9, M10, M42, M45, M53, M54, M55, M62, M63, M65, M66, M67, M68, M69,M71, and M83 bind overlapping epitopes on OSM and display in vitroand/or in vivo OSM inhibiting activities. Significantly, the reactivityof the selected MAbs includes the ability to dose-dependently block OSMinteraction with gp130, reduce OSM signaling in the presence of gp130,reduce OSM-stimulated proliferation of A375 cells, preventmacrophage-stimulated chondrocyte collagen production, or reducecytokine release by OSM in vivo.

Given the properties of the monoclonal antibodies as described in thepresent invention, the antibodies or antigen binding fragments thereofare suitable both as therapeutic and prophylactic agents for treating orpreventing OSM-associated conditions in humans and animals.

In general, use will comprise administering a therapeutically orprophylactically effective amount of one or more monoclonal antibodiesor antigen binding fragments of the present invention, or an antibody ormolecule selected to have similar spectra of binding and biologicactivity, to a susceptible subject or one exhibiting a condition inwhich OSM activity is known to have pathological sequelae, such asimmunological disorders or tumor growth and metastasis. Any active formof the antibody can be administered, including Fab and F(ab′)2fragments.

Preferably, the antibodies used are compatible with the recipientspecies such that the immune response to the MAbs does not result in anunacceptably short circulating half-life or induce an immune response tothe MAbs in the subject. The MAbs administered may exhibit somesecondary functions, such as binding to Fc receptors of the subject andactivation of ADCC mechanisms, in order to deplete the target cellpopulation using cytolytic or cytotoxic mechanisms or they may beengineered to by limited or devoid of these secondary effector functionsin order to preserve the target cell population.

Treatment of individuals may comprise the administration of atherapeutically effective amount of the antibodies of the presentinvention. The antibodies can be provided in a kit as described below.The antibodies can be used or administered as a mixture, for example, inequal amounts, or individually, provided in sequence, or administeredall at once. In providing a patient with antibodies, or fragmentsthereof, capable of binding to OSM, or an antibody capable of protectingagainst OSM in a recipient patient, the dosage of administered agentwill vary depending upon such factors as the patient's age, weight,height, sex, general medical condition, previous medical history, etc.

In a similar approach, another therapeutic use of the monoclonalantibodies of the present invention is the active immunization of apatient using an anti-idiotypic antibody raised against one of thepresent monoclonal antibodies. Immunization with an anti-idiotype whichmimics the structure of the epitope could elicit an active anti-OSMresponse (Linthicum, D. S. and Farid, N. R., Anti-idiotypes, Receptors,and Molecular Mimicry (1988), pp 1-5 and 285-300).

Likewise, active immunization can be induced by administering one ormore antigenic and/or immunogenic epitopes as a component of a vaccine.Vaccination could be performed orally or parenterally in amountssufficient to enable the recipient to generate protective antibodiesagainst this biologically functional region, prophylactically ortherapeutically. The host can be actively immunized with theantigenic/immunogenic peptide in pure form, a fragment of the peptide,or a modified form of the peptide. One or more amino acids, notcorresponding to the original protein sequence can be added to the aminoor carboxyl terminus of the original peptide, or truncated form ofpeptide. Such extra amino acids are useful for coupling the peptide toanother peptide, to a large carrier protein, or to a support Amino acidsthat are useful for these purposes include: tyrosine, lysine, glutamicacid, aspartic acid, cysteine and derivatives thereof. Alternativeprotein modification techniques may be used, e.g., NH2-acetylation orCOOH-terminal amidation, to provide additional means for coupling orfusing the peptide to another protein or peptide molecule or to asupport.

The antibodies capable of protecting against unwanted OSM bioactivityare intended to be provided to recipient subjects in an amountsufficient to effect a reduction, resolution, or amelioration in theOSM-related symptom or pathology. An amount is said to be sufficient ora “therapeutically effective amount” to “effect” the reduction ofsymptoms if the dosage, route of administration, etc. of the agent aresufficient to influence such a response. Responses to antibodyadministration can be measured by analysis of subject's affectedtissues, organs, or cells as by imaging techniques or by ex vivoanalysis of tissue samples. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient.

Therapeutic Applications

The OSM-neutralizing antibodies of the present invention, antigenbinding fragments, or specified variants thereof can be used to measureor cause effects in an cell, tissue, organ or animal (including mammalsand humans), to diagnose, monitor, modulate, treat, alleviate, helpprevent the incidence of, or reduce the symptoms of, a conditionmediated, affected or modulated by OSM or cells expressing OSM. Thus,the present invention provides a method for modulating or treating atleast one OSM related disease, in a cell, tissue, organ, animal, orpatient, as known in the art or as described herein, using at least oneOSM antibody of the present invention.

OSM is known to be up-regulated in a variety of disease states thatinvolve inflammation and has been implicated in diverse biological rolesincluding bone formation, cartilage degradation, cholesterol uptake,pain, and inflammation. Particular indications are discussed below.

Indications

The present inventors have demonstrated that OSM mediates cartilagedestruction and shown that OSM causes chondrocyte degradation inintra-articular matrix from tissue explants. OSM also promotes cytokinerelease, such as TNFα, which is able to promote collagen release fromcartilage as shown by T. Cawston et al (1998, Arthritis and Rheumatism,41(10) 1760-1771) and that presently an antibody exemplified as M71binding domains is capable of blocking systemic cytokine release. Anantibody of the present invention; M55, M64, M69, and M71 demonstratedthe ability to increase proteoglycan synthesis in amacrophage-chondrocyte co-culture system above the level seen in theabsence of an OSM-specific antibody.

The present inventors have further demonstrated that administration of aneutralising anti-OSM antibody of the invention will inhibit OSM drivencytokine and chemokine release in vivo, such as IL-6, IP-10, and KC.IP-10, interferon gamma-induced protein 10 kDa or small-induciblecytokine B10, is a protein that in humans is encoded by the CXCL10 gene(C-X-C motif chemokine 10 (CXCL10). CXCL10 has been attributed toseveral roles, such as chemoattraction for monocytes/macrophages, Tcells, NK cells, and dendritic cells, and promotion of T cell adhesionto endothelial cells. KC, now known as chemokine (C-X-C motif) ligand 1(CXCL1), is a small cytokine belonging to the CXC chemokine family thatwas previously called GRO1 oncogene, GROα, Neutrophil-activating protein3 (NAP-3) and melanoma growth stimulating activity, alpha (MSGA-α). Inhumans, this protein is encoded by the CXCL1 gene. CXCL1 is expressed bymacrophages, neutrophils and epithelial cells, and has neutrophilchemoattractant activity.

According to the present invention there is therefore provided the useof an antibody or antibody fragment selected from the group M5, M6, M9,M10, M42, M45, M53, M54, M55, M62, M63, M65, M66, M67, M68, M69, M71,and M83 in the manufacture of a medicament for the treatment orprophylaxis of an articular proteoglycan degradative disease such asosteoarthritis, an inflammatory arthropathy or inflammatory disorder. Aparticular use of an antagonist of OSM is in the manufacture of amedicament to prevent or reduce collagen release from cartilage. Theinvention further provides a method for the treatment or prophylaxis ofan inflammatory arthropathy or inflammatory disorder comprisingadministering an effective amount of such an antibody which blocks OSMbinding to gp130 to a patient suffering from such a disorder.

An antibody of the invention may used in a preparation for the treatmentof pro-inflammatory process in which OSM directly or indirectly, such asthrough the release of inflammatory cytokines, leads to pathogenesis intissues or organs especially in the skin, lungs, and joints. Suchpathologies include osteoarthritis, rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, neuropathic arthropathy, reactivearthritis, rotator cuff tear arthropathy, rheumatic fever, Reiter'ssyndrome, progressive systemic sclerosis, primary biliary cirrhosis,pemphigus, pemphigoid, necrotizing vasculitis, myasthenia gravis,multiple sclerosis, lupus erythematosus, polymyositis, sarcoidosis,granulomatosis, vasculitis, pernicious anemia, CNS inflammatorydisorder, antigen-antibody complex mediated diseases, autoimmunehaemolytic anemia, Hashimoto's thyroiditis, Graves disease, Reynard'ssyndrome, glomerulonephritis, dermatomyositis, chronic active hepatitis,celiac disease, autoimmune complications of AIDS, atrophic gastritis,and Addison's disease, endotoxemia or septic shock (sepsis), or one ormore of the symptoms of sepsis and other types of acute and chronicinflammation. Those patients who are more particularly able to benefitfrom the method of the invention are those suffering from infection byE. coli, Haemophilus influenza B, Neisseria meningitides, staphylococci,or pneumococci. Patients at risk for sepsis include those suffering fromburns, wounds, renal or hepatic failure, trauma, burns,immunocompromised (HIV), hematopoietic neoplasias, multiple myeloma,Castleman's disease or cardiac myxoma.

Other conditions that are associated with OSM and amenable to treatmentor preventative therapy with the antibodies of the invention includefibrotic disease such as pulmonary fibrosis, diabetic nephropathy,idiopathic pulmonary fibrosis, systemic sclerosis, and cirrhosis.Another indication for use of antibodies of the invention is in thetreatment or prevention of nociceptive pain involving neurons of dorsalroot ganglia.

Administration and Dosing

The invention provides for stable formulations of an OSM-neutralizingantibody, which is preferably an aqueous phosphate buffered saline ormixed salt solution, as well as preserved solutions and formulations aswell as multi-use preserved formulations suitable for pharmaceutical orveterinary use, comprising at least one OSM-neutralizing antibody in apharmaceutically acceptable formulation. Suitable vehicles and theirformulation, inclusive of other human proteins, e.g., human serumalbumin, are described, for example, in e.g. Remington: The Science andPractice of Pharmacy, 21st Edition, Troy, D. B. ed., Lipincott Williamsand Wilkins, Philadelphia, Pa. 2006, Part 5, PharmaceuticalManufacturing pp 691-1092, See especially pp. 958-989.

The OSM-neutralizing antibody in either the stable or preservedformulations or solutions described herein, can be administered to apatient in accordance with the present invention via a variety ofdelivery methods including intravenous (I.V.); intramusclular (I.M.);subcutaneously (S.C.); transdermal; pulmonary; transmucosal; using aformulation in an implant, osmotic pump, cartridge, micropump; or othermeans appreciated by the skilled artisan, as well-known in the art.

For example, site specific administration may be to body compartment orcavity such as intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracelebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, intralesional,vaginal, rectal, buccal, sublingual, intranasal, or transdermal means.

In general, if administering a systemic dose of the antibody, it isdesirable to provide the recipient with a dosage of antibody which is inthe range of from about 1 ng/kg-100 ng/kg, 100 ng/kg-500 ng/kg, 500ng/kg-1 ug/kg, 1 ug/kg-100 ug/kg, 100 ug/kg-500 ug/kg, 500 ug/kg-1mg/kg, 1 mg/kg-50 mg/kg, 50 mg/kg-100 mg/kg, 100 mg/kg-500 mg/kg (bodyweight of recipient), although a lower or higher dosage may beadministered. Of course, suitable dosages of an antagonist of thepresent invention will vary, depending upon factors such as the diseaseor disorder to be treated, the route of administration and the age andweight of the individual to be treated and the nature of the antagonist.Without being bound by any particular dosages, it is believed that forinstance for parenteral administration, a daily dosage of from 0.01 to20 mg/kg of an antibody (or other large molecule) of the presentinvention (usually present as part of a pharmaceutical composition asindicated above) may be suitable for treating a typical adult.

The treatment may be given in a single dose schedule, or preferably amultiple dose schedule in which a primary course of treatment may bewith 1-10 separate doses, followed by other doses given at subsequenttime intervals required to maintain and or reinforce the response, forexample, at 1-4 months for a second dose, and if needed, a subsequentdose(s) after several months. Examples of suitable treatment schedulesinclude: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii)0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient toelicit the desired responses expected to reduce disease symptoms, orreduce severity of disease.

The antibodies of the present invention may be used alone or incombination with immunosuppressive agents such as steroids (prednisoneetc.), cyclophosphamide, cyclosporin A or a purine analogue (e.g.methotrexate, 6-mercaptopurine, or the like), or antibodies such as ananti-lymphocyte antigen antibody, an anti-leukocyte antigen antibody, aTNF antagonist e.g. an anti-TNF antibody or TNF inhibitor e.g. solubleTNF receptor, or agents such as NSAIDs or other cytokine inhibitors.

SEQUENCE TABLE SEQ ID NO: Description SEQUENCE AND FEATURES 1Human IGHV1- QVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYX ₁ ISWVRQA 69*01PGQGLEWMGX ₂  IX ₃ X ₄ X ₅ X ₆ GTANY AQKFQGRVTI TADESTSTAYMELSSLRSED TAVYYCAR Where X₁ is A or G, X₂ is G or W, X₃ may be Ior S, X₄ may be P or A, X₅ may be I or Y and X₆ may be F or N. 2Human IGHV3- EVQLLESGGG LVQPGGSLRL SCAASGFTFS X ₁ YX ₂ MX ₃WVRQA 23*01PGKGLEWVSX₄  IX ₅ X ₆ X ₇ GX ₈ STYY ADSVKGRFTI SRDNSKNTLYLQMNSLRAED TAVYYCAK Where X₁ may be S, D, N, or T; X₂ may be A, G,or W; X₃ may be S or H; X₄ may by V, A, N or G;X₅ may be S, N, K or W; X₆ may be Y, S, G, or Q;X₇ may be S or D; _(and) X₈ may be S or G 3 Human IGHV5-EVQLVQSGAE VKKPGESLKI SCKGSGYSFT X ₁ YWIX ₂WVRQM 51*01 PGKGLEWMGX ₃ IX ₄PX ₅ DSX ₆ TRY SPSFQGQVTI SADKSISTAY LQWSSLKASD TAMYYCARWhere X₁ may be S, N, or T; X₂ may be S or G; X₃may be I or R; X₄ may by D or Y; X₅ may be G or S; X₆, may be D or Y 4Human JH WGQGTLVTVSS 5 Human IGKV1- DIQMTQSPSS LSASVGDRVT ITCRASQSI X₁ X ₂ X ₃ LNWYQQKP 39*01 (O12) GKAPKLLIYX ₄ ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCWhere X₁ may be A, D, N, S, or R; X₂ may be D,G, K, N, or S; X₃ maybe A, D, F, H, N, S, W,V, or Y; X₄ may be A, D, F, G, K, N, T, or Y. 6 IGKV3-11 (L6)EIVLTQSPAT LSLSPGERAT LSCRASQSV X ₁ X ₂ X ₃ LAWYQQKP GQAPRLLIYX₄ ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCWhere X₁ may be A, D, N, R or S; X₂ may be D,K, N, or S; X₃ may be A, D, F, H, N, S, W, orY; X₄ may be A, D, K, G, F, T or N. 7 Human IGKV3-EIVLTQSPGT LSLSPGERAT LSCX ₁ X ₂ X ₃ X ₄ SVS SSYLAWYQQK 20 (A27)PGQAPRLLIY X ₅ ASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCWhere X₁ may be D, N, S, R or T; X₂ may be N, S,or R; X₃ may be A, D, H, N, S, or R; X₄ may beE, F, H, K, Q, S, or Y; X₅ may be A, D, G, or S. 8 Human IGKV4-DIVMTQSPDS LAVSLGERAT INCKSSQSVL X ₁ SSNNX ₂ NX ₃ LA 1*01 (B3)WYQQKPGQPP KLLIYX ₄ ASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCWhere X₁ may be A, F, H, S, or Y; X₂ may be E,K, N, or T; X₃ maybe A, D, F, H, N, S, W, orY; X₄ may be A, D, S, W or Y. 9 L-CDR3 QQX₁X₂X₃X₄PX₅T formulaWhere X₁ may be A, D, F, H, P, S, or Y; X₂ maybe D, E, G, H, I, K, N, R, T or Y; X₃ may beD, G, H, K, N, S, T, or R; X₄ may be F, I, L,N, R, W or Y as defined in Table 2. 10 JK4 FGQGTKVEIK 11 human OSMAAIGSCSKEYRVLLGQLQKQTDLMQDTSRLLDPYIRIQGLDVPKLR protein EHCR sequenceERPGAFPSEETLRGLGRRGFLQTLNATLGCVLHRLADLEQRLPKAQ DLERSGLNIEDLEKLOMARPNILGLRNNIYCMAOLLDNSDTAEPTKAGRG ASAPPTPTPASDAFQRKLEGCRFLHGYHRFMHSVGRVFSKWGESPNRSRR HSPHQALRKGVRRTRPSRKGKRLMTRGQLPR 12 Cyno (MacacaAAMGSCSKEYRMLLGQLQKQTDLMQDTSRLLDPYIRIQGLDIPKLR fascicularis) EHCROSM protein ESPGAFPSEETLRGLGRRGFLQTLNATLGRVLHRLADLEQHLPKAQ DLERSGLNIEDLEKLQMARPNVLGLRNNIYCMAQLLDNSDMTEPTKAGRG TPQPPTPTPTSDVFQRKLEGCSFLRGYHRFMHSVGRVFSKWGESPNRSRR HSPHQALRKGVRRTRPSRKGNRLMPRGQLPR 13 H2 H-CDR1 SYAIS 14 H14 & H17 H- SYWISCDR1 15 H135 H-CDR1 SYWIG 16 H2 H-CDR2 GIIPIFGNANYAQKFQG 17 H14 & H17 H-IIYPGDSYTRYSPSFQG CDR2 18 H135 H-CDR2 IIYPGDSDTRYSPSFQG 19 H2 H-CDR3YGAKGLLDY 20 H14 H-CDR3 GSVFEAYFDY 21 H17 H-CDR3 VPVSPAYLDY 22H135 H-CDR3 GFGASYLDY 23 B3 & L2 L- KSSQSVLYSSNNKNYLA CDR1 24 L12 L-CDR1KSSQSVLSSSNNENWLA 25 L111 L-CDR1 KSSQSVLASSNNNNFLA 26 B3 L-CDR2 WASTRES27 B3 L-CDR3 QQYYSTPL 28 L2 L-CDR3 QQSFSFPI 29 L-CDR3QQ-(SY)-(FY)-S-(FT)-PLT consensus 30 L171-CDR1 KSSQSVLSSGNNGNYLA 31L172-CDR1 KSSQSVLSSGSNHNYLA 32 L173-CDR1 KSSQSVLSSRGNNNYLA 33 L174-CDR1KSSQSVLGSWGNDNYLA 34 L175-CDR1 KSSQSVLYSGGNGNYLA 35 L176-CDR1KSSQSVLGSWGNGHYLA 36 L177-CDR1 KSSQSVLSSNGNHNYLA 37 L178-CDR1KSSQSVLSSDGNHNYLA 38 L180-CDR1 KSSQSVLGSSSNINFLA 39 L182-CDR1KSSQSVLGSGDNRNYLA 40 L184 & L186 KSSQSVLGSGYNRNYLA L-CDR1 41 L192 L-CDR1KSSQSVLGSWHNDNYLA 42 L-CDR2 KASTRES 43 L180-CDR2 SASTRES 44 L182-CDR2NASTRES 45 L175-CDR3 QQYSTTPLT 46 L180-CDR3 QQYFSTPIT 47 Reselected L-QQY-(F,Y)-STP-(L,I)-T CDR3 consensus 48 H- FR1-CDR1-EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWISWVRQMPGKGLE FR2-CDR2-FR3WMGIIYPGDSYTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTA MYYC 49 L173DIVMTQSPDSLAVSLGERATINCKSSQSVLSSRGNNNYLAWYQQKPGQPPKLLIYKASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQQYYSTPL 50 L178DIVMTQSPDSLAVSLGERATINCKSSQSVLSSDGNHNYLAWYQQKPGQPPKLLIYKASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQQYYSTPL 51 L180DIVMTQSPDSLAVSLGERATINCKSSQSVLGSSSNINFLAWYQQKPGQPPKLLIYSASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQQYFSTPI 52 L185DIVMTQSPDSLAVSLGERATINCKSSQSVLSSGGNWNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQQYYTTPL 53 L186DIVMTQSPDSLAVSLGERATINCKSSQSVLSSGSNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQQYYSTPL 54 H14EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWISWVRQMPGKGLEWMGIIYPGDSYTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTA MYYCARGSVFEAYFDY 55 H17EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWISWVRQMPGKGLEWMGIIYPGDSYTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTA MYYCARVPVSPAYLDY 56AviTag GLNDIFEAQKIEWHE

The present invention will now be described with reference to thefollowing specific, non-limiting examples.

Example 1 Reagents and Assays

In order to select and characterize OSM-binding antibodies, constructsof human and Cyno OSM were generated for mammalian cell expression.Human OSM (NP_(—)065391 encoded by NM_(—)020530) is a 252 amino acidprecursor processed into a full-length secreted protein of 227 aminoacids (SEQ ID NO: 11), which is a proprotein further processed into themore fully active mature form, amino acids, 1-184. Human OSM cDNA wasordered from OriGene (Cat. No. SC121421) and the ORF of human OSM fromthe OriGene clone was amplified by PCR and a signal peptide (murineIgG1) was introduced along with a hexa-His tag for protein purificationand an AviTag (SEQ ID NO: 56) for site-directed protein biotinylation.The latter was chosen to avoid random chemical biotinylation of lysineresidues present in the vicinity of OSM intereaction with receptors.

Cynomolgous monkey OSM was cloned from cyno PBMC's RNA using SuperscriptIII first strand synthesis system (InVitrogen) to obtain the cDNA andthen PCR amplified using the UTR primers designed from the human OSMsequence as described in U.S. patent application Ser. No. 12/648,430.The expressed full-length protein is shown in SEQ ID NO: 12 with thecleaved, active form represented by 184 residues, 51-227.

Precursor and mature forms of human and Cyno OSM were expressed in HEK293 and purified using standard methodologies. The functional activitiesof the proteins were tested in the A375-S2 cell proliferation and pSTAT3signaling assays using as control E. coli derived commercially availablehuman OSM (R&D Systems, Cat. No. 295-OM).

Mabs

Where a control antibody was used, a human IgG1 isotype antibodydesignated CNTO6234 was used.

Chemical Biotinylation

Recombinant human OSM was biotinylated using NHS-ester chemistry(EZ-Link Sulfo-NHS-LC-Biotinylation Kit, Pierce, #21435) targeting amineresidues on the cytokine. The biotin-coupling reaction was optimized fora target labeling efficiency of one mole of biotin per mole of antigen.The latter minimizes the loss of binding and functional activity whileensuring near-complete labeling of the protein population. Uponcompletion of the reaction, the protein was purified from the freebiotin reagent and residual leaving groups using Zeba Desalt SpinColumns included in the EZ-Link Sulfo-NHS-LC-Biotinylation Kit (Pierce).Approximately 80% of the starting material was recovered. The HABA assay(Pierce Biotin Quantitation Kit, #28005) was used to measure the levelof biotin incorporation, which indicated approximately one mole ofbiotin per mole of human OSM. An Octet instrument (FortéBIO) was used toverify both streptavidin coupling and gp130-Fc (R&D Systems, Cat. No.671-GP) binding for the biotinylated protein. Octet measurements showedthat biotinylated human OSM bound gp130 with a profile essentiallyidentical to that of the unlabeled starting material.

In Vitro Targeted Biotinylation

The 15 residue AviTag (GLNDIFEAQKIEWHE) (SEQ ID NO: 56) has similarbiotin-acceptor kinetics as the endogenous BirA substrate BCCP (Beckettet. al. 1999, Protein Science). When tethered to a protein of interestthe AviTag, having one acceptor lysine residue, will be biotinylated atonly one position. Recombinant Cyno OSM was biotinylatedsite-specifically in vitro using biotin-protein ligase and reagentscommercially available from Avidity. The biotinylated Cyno OSM waspurified using monovalent streptavidin affinity resin. The quality ofthe resulting protein was assessed by SDS-PAGE and SEC-HPLC. The HABAassay (Pierce Biotin Quantitation Kit, #28005) was used to measure thelevel of biotin incorporation, which indicated approximately one mole ofbiotin per mole of cyno OSM. An Octet instrument (FortéBIO) was used toverify both streptavidin coupling and gp130-Fc chimera binding for thebiotinylated protein. Octet measurements showed that biotinylated CynoOSM bound gp130 with a profile essentially identical to that of theunlabeled starting material.

Solid Phase Immunoassays

Initial phage-Fab panning using NEUTRAVIDIN™ ELISA plates (Pierce)coated with 2 μg/ml biotinylated human OSM or cyno OSM in TBS. Afterovernight incubation at 4° C., blocking and washing, 1:100 dilutions ofpolyclonal phage pools from each round of panning were added. Boundphage was detected with an HRP-conjugated monoclonal specific for pVIII,the M13 phage major coat protein (GE Healthcare, Cat. No. 27-9421-01),followed by addition of chemiluminescent substrate POD (Roche, Cat. No.11582950001), and read on a PerkinElmer instrument.

Primary screening of individual clones was performed using secretedsoluble Fab-His protein from E. coli supernatants. Bacterialsupernatants, containing soluble Fab-His protein, were used to carry outbinding in an ELISA formats. Black MaxiSorp plates (Nunc, Cat. No.437111) were coated with 1 μg/ml sheep anti-human Fd (CH1) antibody (TheBinding Site, Cat. No. PC075) and incubated at 4° C. overnight. Afterthe plates were washed and blocked, 50 μl undiluted bacterialsupernatant (containing Fab-His protein) was added and allowed toincubate for 1 hour at room temperature with gentle shaking. Plates werewashed and biotinylated human or Cyno OSM 20 nM was added to thecaptured Fabs. After 1 hour at room temperature, SA-HRP (Invitrogen,Cat. No. 43-4323) was added and chemiluminescent detection carried outas above. It was calculated that primary binding ELISA screening withhuman OSM at a concentration of 20 nM would allow the detection ofclones with affinities in the nanomolar affinity range.

Epitope Binning

Epitope binning is a competition assay performed in order to group theMAbs on the basis of binding characteristics performed using human IgG1converted mAbs and the commercial antibody, MAB29, which is known to bean OSMRβ/LIFRα recruitment-blocker (R-blocker).

To each well of a 384-well multi-array plate (Meso Scale Discovery(MSD), L25XA-4) was added 2.5 μg/ml anti-human Fc (Jackson Immuno,709-005-149) in phosphate buffered saline (PBS) pH7.4 (Sigma, P3813).The plate was incubated at 4′C overnight then blocked with 50 μl of MSDblocker A at room temperature for 1 hr. The 384 multi-array plate waswashed three times (PBS pH7.4, 0.05% Tween 20 (Scytek, PBT010) and toeach well was added a 1.0 μg/ml solution of test mAb followed by shakingat level 6 on a titer plate shaker at room temperature for 1 hr. Theplate was washed three times as before.

In parallel, a 5 μg/ml solution of a competing mAb and 1 μg/mlbiotinylated OSM (R&D Systems, 295-OM-010/CF) were combined in aseparate 96-well plate (COSTAR, 3357) in MSD assay buffer (1:3 of blockbuffer with PBS pH7.4, 0.05% Tween 20) and shaken at level 3 on a titerplate shaker at room temperature for 1 hr. The pre-complex of competingantibody and biotinylated OSM was added to each well of the 384multi-array plate and shaken (level 6 on a titer plate shaker) at roomtemperature for 1 hr. The plate was washed three times and to each wellwas added streptavidin sulfo TAG (MSD, R32Ad-S) and shaken (level 6 on atiter plate shaker) at room temperature for 30 min. The plate was washedthree times and to each well was added read buffer T diluted 1:4 withdistilled water (MSD, R92TC-1). The plate was read on an MSD 56000instrument.

The data was interpreted on the basis of the signal attained for a testmAb in the presence of biotinylated OSM with no competing mAb present(maximum signal) together with the signal from self competition (2-4fold reduction in maximum signal). Epitope competition was assigned whenthe value in the presence of a competing mAb was within three standarddeviations of the value of that from self competition.

A375 Cells

A375 cells (ATCC; CRL-1619) are a human epithelia cell derived frommalignant melanoma. A375-S2 cells (ATCC; CRL-1872, a subline of CRL-1619sensitive to IL-1) are cultured in complete growth medium(DMEM/GlutaMax-I, Gibco) supplemented with 10% FBS (Gibco) in T-175culture flasks (Corning; Cat. No. 431080) and sub-cultured whenapproximately 80% confluent every three to four days in a 1:20sub-culture ratio.

A375-S2 Cell Proliferation (BrdU-Incorporation) Assay

The reduction in A375-S2 cells proliferation by human or Cynomolgusmonkey OSM was measured by BrdU-incorporation using a chemiluminescentELISA. Oncostatin M reduces proliferation in the A375-S2 human melanomacell line (Zarling et al. PNAS 83:9739-9743). This cell line was used toevaluate the anti-oncostatin M antibodies at all stages of antibodydiscovery. A375-S2 cells were maintained in T-150 tissue culture flasks(BD Falcon 35-5001) in DMEM (Gibco 11995)+10% FBS (Gibco 16140)+1%Pen/Strep (Gibco 15140) at 37° C. in 95% O2—5% CO2 and split 1:10 twiceper week.

To perform the proliferation assay, cells were trypsinized (0.25%, Gibco25200) to remove them from the T150 flasks and then plated at a densityof 2000 cells/well in the inner 48 wells of black TC-coated plates (BDFalcon 353948) in 200 μL of DMEM/FBS/Pen-Strep. After overnightincubation at 37° C./95% O2—5% CO2, the media was removed and replacedwith 180 μL of fresh media. A separate plate was prepared whichcontained all of the test solutions at 10× the final concentrations.From this plate, 20 μL was transferred to the corresponding well in thecell plate to generate the appropriate experimental conditions. Eachexperimental condition was tested in triplicate. In any experiment whichevaluated neutralization, the antibody and Oncostatin M were incubatedtogether for at least 1 hour being added to cells. The plates were thenincubated at 37° C./95% O2—5% CO2 for an additional 72 hours. At thistime the Chemiluminescent BrdU Cell Proliferation ELISA (Roche11669915001) was performed. The BrdU labeling reagent was added to theculture for 4 hours. The media was then removed, and 100 μL of fixsolution was added to each well. After 30 min at room temperature thesolution was removed and 100 μL/well of anti-BrdU-POD solution wasadded. After 2 hours at room temperature, the plate was washed withPBS-Tween and 100 μL of Super Signal Pico (Thermo Scientific 37069) wasadded to each well. Luminescence was then read using a Perkin ElmerVictor3 instrument.

Initial experiments were done to determine the dose-response of thein-house generated CHO cell-derived recombinant human and cynomolgousmonkey oncostatin M in suppressing proliferation. Osm was tested from astarting concentration of 100 ng/ml with 1 to 5 dilutions to a finalconcentration of 0.0244 ng/ml. Percent inhibition of proliferation wascalculated compared to cells treated with vehicle control. The resultsof such an experiment are shown in FIG. 2A. As the concentration ofhuman or cyno Oncostatin M is increased there is a correspondingreduction in BrdU incorporation. The antiproliferative activity of humanand cynomolgous monkey Oncostatin M is indistinguishable, based on boththe extent of anti-proliferative activity and on the EC50 of the effect.

Based on these experiments, a concentration of 2 ng/ml of Oncostastin Mwas used to evaluate and compare anti-Oncostatin M antibodies becausethis concentration inhibited proliferation approximately 80%, giving alarge window in which to determine antibody dose responses. A fulldose-range of antibody was evaluated in the presence of 2 ng/ml of humanor cyno Oncostatin M. In addition, an isotype control was included inthe experiment at the highest concentration of anti-Oncostatin Mantibody used. The assay window was defined by the difference betweenuntreated control wells (maximum proliferation) and wells incubated with2 ng/ml Oncostatin M alone (minimum proliferation) and the percentneutralization at any concentration of antibody was defined within thiswindow.

pSTAT3 Signaling Using A375-S2 Phospho-Stat3Assay

OSM inhibits the growth of A375-S2 cells by binding cell surfacereceptor gp130 and inducing receptor heterodimerization with OSMRβ toinitiate an intracellular signaling cascade that includes activation(phosphorylation) of the signaling molecule STAT3 (Kortylewski et al.,Oncogene 18: 3742-3753, 1999). Disruption of STAT3 signaling abolishesOSM growth inhibition of A375-S2 cells, demonstrating that STAT3activation is a key step in OSM signaling (Heinrich et al., Biochem. J.374: 1-20, 2003). STAT3 phosphorylation in A375-S2 cells has been shownto be OSM concentration dependent and commercial kits are available tomeasure it in stimulated cells. Neutralization of OSM-induced STAT3phosphorylation in A375-S2 cells was chosen as a primary screening assayfor Mabs candidates.

For antibody neutralization of OSM-induced STAT3 phosphorylation A375-S2cells are seeded into 96-well tissue culture plates (Corning; Cat. No.3596) at 25,000 cells/well in 200 μl in complete growth media andincubated for 24 hours. Cells are treated with a solution containing 5ng/ml human OSM (recombinant, mammalian-cell derived, OSMN1-1) which hasbeen pre-incubated for 3 hours at room temperature with 1:5 seriallydiluted experimental mAb starting at 10 μg/ml. Controls includeuntreated cells, stimulated cells (5 ng/ml OSM only) and a hIgG1 isotypecontrol mAb. All treatments are performed in triplicate unless otherwisenoted.

Analysis of pSTAT3 content is carried out using the Phospho-STAT3Whole-Cell Lysate Kit (MSD; Cat. No. K150DID-1, Lot No. K0010570)following the manufacturer's protocol. Briefly, the cells are treated ina 200 μl/well volume for 10 minutes; treatment solutions are removed and50 μl of cell MSD lysis buffer is added via multichannel pipette. Theplate is placed on an orbital shaker (at 300 RPM) for 5 minutes.Thereafter, 30 μl of each lysate is transferred to the MSD phospho-STAT396-well plate. The plate is sealed and placed on an orbital shaker (at300 RPM) for 1 h at room temperature, washed three times with 150 μl ofMSD wash buffer, and 25 μl of secondary detection antibody conjugate(anti-pSTAT3-Ru(bpy)32+) added to each well, again sealed and incubatedon an orbital shaker (at 300 RPM) for 1 h at room temperature. The plateis washed as previously and 150 μl of MSD read buffer (tripropylaminesolution) is added to each well. The plate is read on an MSD SECTORImager 6000 instrument.

Full EC50 dose-response curves for inline matured, parental and controlmAbs were obtained and plotted as normalized percent pSTAT3 signal.

Affinity Measurement by Surface Plasmon Resonance (Biacore)

The binding affinities were measured using Surface Plasmon Resonance(SPR) with a Biacore 3000 optical biosensor (Biacore) using human orCyno OSM constructs as described. A biosensor surface was prepared bycoupling anti-IgG Fc antibody mixture of anti-Mouse (Jackson, Cat. No.315-005-046) and anti-Human (Jackson, Cat. No. 109-005-098) to thecarboxymethylated dextran surface of a CM-5 chip (Biacore, Cat. No.BR-1000-14) using the manufacturer's instructions for amine-couplingchemistry. Approximately 19,000 RU (response units) of anti-OSM antibodywere immobilized in each of four flow cells. The kinetic experimentswere performed at 25° C. in running buffer (DPBS+0.005% P20+3 mM EDTA).Serial dilutions Human and Cyno OSM ECD from 100 nM to 0.412 nM wereprepared in running buffer. About 200 RU of mAb were captured on flowcells 2 to 4 of the sensor chip. Flow cell 1 was used as referencesurface. Capture of mAb was followed by a three-minute injection(association phase) of antigen at 50 μl/min, followed by 10 minutes ofbuffer flow (dissociation phase). The chip surface was regenerated bytwo pulses of 18-second injections of 100 mM H3PO4 (Sigma, Cat. No.7961) at 50 μl/min.

The collected data was processed using the BIAevaluation software(Biacore, version 3.2). First, double reference subtraction of the datawas performed by subtracting the curves generated by buffer injectionfrom the reference-subtracted curves for analyte injections. Kineticanalysis of the data was performed using 1:1 binding model with globalfit. The result for each mAb was reported in the format of Ka (On-rate),Kd (Off-rate) and K_(D) (affinity constant).

Example 2 Selection Of OSM Binding Fabs

The de novo Fab-pIX libraries have been described Shi et al. J Mol Biol397:385-396, 2010; WO09085462A1; U.S. Ser. No. 12/546,850; and hereinabove are designated 169, 323 and 551 which references the heavy-chainhuman germline framework being used: IGHV1-69 (SEQ ID NO: 1), IGHV3-23(SEQ ID NO: 2), or IGHVS-51(SEQ ID NO: 3) in IMGT nomenclature. Thethree heavy-chain library frameworks are combined with four light-chainlibrary VL_(kappa) frameworks: A27 (IGKV3-20*01 (SEQ ID NO: 5)), B3(IGKV4-1*01 (SEQ ID NO: 6)), L6 (IGKV3-11*01 (SEQ ID NO: 7)), and 012(IGKV1-39*01 (SEQ ID NO: 8)). In the libraries, the Fabs V-regions arecompleted by the addition of a J-region (FR4) comprising SEQ ID NO: 4 inthe heavy chains and SEQ ID NO: 10 in the light chains. The heavy chainCDR3 is of variable length from 7-14 residues. Examples of the completeV-regions for each library are shown in FIG. 1 and numbered and CDRregions shown according to Kabat.

The initial set of anti-OSM phage display hits were identified usingcommercially available aglycosylated human OSM. The Fab-pIX phagedisplay libraries were panned using biotinylated human OSM (R&D Systems,Cat. No. 295-OM) capture on paramagnetic Streptavidin (SA) beads(Invitrogen, Cat. No. 112.05D) following a published protocol for phageselection (Marks and Bradbury, Antibody Engineering, Vol. 248: 161-176,Humana Press, 2004). Briefly, biotinylated human OSM was added to thephage libraries that had been pre-absorbed on unconjugated beads, to afinal concentration of 100 nM and incubated for 1 hour with gentlerotation. Blocked SA beads were added and incubated for 15 minutes tocapture biotinylated OSM with bound phage. The magnetically-capturedphage/antigen/bead complex was washed 5 times with 1 ml of TBST and oncewith 1 ml TBS. Following removal of the final TBS wash, 1 ml ofexponentially growing TG1 cells (Stratagene, Cat. No. 200123) was addedand incubated at 37° C. for 30 minutes without shaking. Infectedbacteria were spread on LB/Agar (1% Glucose/100 mg/ml Carbenicillin)plates (Teknova, Cat. No. L5804) and incubated overnight at 37° C.Bacterial lawns were scraped and glycerol stocks prepared [15%glycerol/Carbenicillin (100 mg/ml)/2×YT] and stored at −80° C. Toprepare phage for second-round panning, 25 ml of 2×YT/Carbenicillin (100μg/ml) was inoculated with 25 μl of bacterial glycerol stock and grownat 37° C. until an OD600 of roughly 0.5. Helper phage VCSM13(Stratagene, Cat. No. 200251) was added to the culture at a multiplicityof infection of approximately 10:1 and incubation was carried out for 30minutes at 37° C. without shaking. The bacteria was spun down and thepellet resuspended in induction media (2×YT/Carb/Kan/IPTG) and grown at30° C. overnight. Phage was precipitated with 2% PEG/0.25M NaCl (finalconcentrations) and re-suspended in 2 ml of PBS. First-round phage wasstored at 4° C. and used to carry out second-round panning. The panningparameters were: Round 1, 100 nM antigen, 1 hour incubation at roomtemperature, 5× washes with TBST followed by 1× wash with TBS; Round 2,10 nM antigen, 1 hour incubation at room temperature, 10× washes withTBST/1× wash with TBS; and Round 3, 1 nM antigen, 16 hour (overnight)incubation at 4° C., 10× washes with TBST/1× wash with TBS.

Success was monitored using an ELISA where Fabs were captured with ananti-human Fd (CH1) antibody and biotinylated human OSM was added at 20nM and bound OSM was detected with SA-HRP.

Thirty (30) unique heavy chain-light chain pairings as displayed Fabswere identified that were shown to cross react with Cyno OSM by ELISA.The heavy chains represented sequences from the 169 (IGHV1-69 derived)and 551 (IGHV5-51 derived) libraries and were combined with light chainvariable regions representing all four of the library germline origins(A27, B3, L6, and 012).

Example 3 Characterization of OSM Binding Mabs

The four-helix bundle architecture OSM is characterized by four αhelical segments designated A, B, C and D linked by relativelyunstructured loops. OSM interacts with gp130 via an surface located inhelices A and C (Site II) which was determined to include contact byamino acid residues Q16, Q20, G120, N123, N124 of SEQ ID NO: 1 (Delleret al. Structure 8(8): 863-874, 2000; Liu et al. Int. J. Mol. Med. 23:161-172, 2009). The surface responsible for OSM interaction with OSMRβand LIFRα (Site III) is believed to be largely defined by residueslocated in helix D (Deller et al. ibid).

It was the objective to select high affinity binders to OSM capable ofpreventing OSM driven gp130 signaling either through the prevention ofOSM binding to gp130 (Site II or B-blocker) or prevention of OSM boundgp130 recruitment of the LIFRα or OSMRb (Site III or R-blocker).

Of the 30 initially selected OSM-binding Fabs, 29 were cloned intovectors for conversion to full-length human IgG1 Mabs. Thecharacterization assays were

(1) competitive binding to identify epitope groups or “bins”, (2)affinity measurements by surface Plasmon resonance (Biacore), and (3)ability to block pSTAT3 signaling. All screens and assays have beencarried out using the mammalian cell produced (glycosylated) human andcyno proteins as described in Example 1.

Results

The data for the affinity measurements for a subset of the mAbs selectedon the basis of their ranking relative to control MAB295 (R&D Systems)in the pSTAT3 and ELISA binding assays is shown in Table 3.

TABLE 3 Affinity of select subset of anti-OSM first round mAbs. Bindingto Human OSM Binding to cyno OSM K_(D) ratio ka (1/Ms) kd (1/s) ka(1/Ms) kd (1/s) Cyno/ mAb 10{circumflex over ( )}4 10{circumflex over( )}−4 KD (nM) 10{circumflex over ( )}4 10{circumflex over ( )}−4 KD(nM) Human MAB295 118.00 21.70 1.85 70.95 141.00 19.75 10.70 M2 54.579.03 1.66 50.23 67.40 13.50 8.10 M6 19.15 1.97 1.03 20.30 2.99 1.48 1.40M22 10.20 3.57 3.52 9.43 101.00 107.00 30.40 M9 6.63 0.81 1.22 7.31 0.540.73 0.60 M21 30.00 12.55 4.17 31.80 14.30 4.50 1.10 M10 6.02 0.63 1.055.33 10.05 18.90 18.10 M7 8.24 1.08 1.31 8.49 0.37 0.43 0.30 M3 39.851.75 0.44 41.95 1.20 0.29 0.70 M19 7.89 0.26 0.34 7.70 2.43 3.15 9.40M25 8.19 5.09 6.21 9.45 71.70 75.80 12.20 M24 Weak No NA Binding M8 4.710.33 0.70 4.98 0.31 0.62 0.90 M4 Weak Weak NA M20 16.60 7.43 4.48 15.3016.20 10.60 2.40 M5 6.59 7.79 11.85 9.22 11.10 12.05 1.00 M17 2.78 0.451.61 2.90 0.77 2.64 1.60 M11 8.64 8.32 9.63 19.65 10.70 5.46 0.60 M276.39 34.95 55.50 Weak NA M18 Weak No NA binding

The results of the binning assay showed that M5, M6 and M9 competed witheach other but not with MAB295. M10 did compete with MAB295. These fourmAbs were also shown by the pSTAT3 assay to be functional neutralizersof gp130 signaling. Therefore, it follows that M10 is a R-blocker andthat M5, M6 and M9 block OSM binding to gp130 (B-blockers).

The target affinity for a therapeutic lead was dictated by the need tocompete decisively the OSM-gp130 interaction whose affinity (K_(D)) hasbeen measured to be approximately 1 nM. Therefore, a target affinity forOSM of 100 pM or lower K_(D) was desired.

Neutralizing MAbs M5, M6 and M9, found to be OSM-gp130 interactionblockers, plus M10 were chosen for inline maturation in order to producederivates that met the 100 pM affinity requirement for a therapeuticcandidate. It was an additional desired property that mAbs bindcynomolgus monkey OSM with an affinity within fivefold that for humanOSM (K_(D) of 500 pM or less).

The compositions of the binding domains for these four Mabs were foundto represent four unique heavy and light chain pairs designated as shownin Table 4.

TABLE 4 Mab HC Library HC ID LC ID M10 169 H2 L2 M6 551 H14 L12 M9 551H17 IGKV4-1 (B3) M5 551 H135 L111

The complete variable region sequences comprise the germline sequencesused to create the phage library as described herein above and,therefore, the fixed residues match the unmutated parent germlineresidues. As such, each V-region is comprised of the designated CDR1 andCDR2 within the scaffold designated as SEQ ID NO: 1-3 or 5-8; followedby CDR3 (Table 3 and 4) and for the light chain variable region,respectively (Table 5 for LC and Table 6 for the HC) followed by aJ-region as SEQ ID NO: 4 for the heavy chain and SEQ ID NO: 10 for thelight chain.

TABLE 5 Frame- work (SEQ SEQ ID SEQ ID SEQ ID VH ID ID NO:) H-CDR1 NO:H-CDR2 NO: H-CDR3 NO: H2 1-69 (1) SYAIS 13 GIIPIFGNANYAQKFQG 16YGAKGLLDY 19 H14 5-51 (3) SYWIS 14 IIYPGDSYTRYSPSFQG 17 GSVFEAYFDY 20H17 5-51 (3) SYWIS 14 IIYPGDSYTRYSPSFQG 17 VPVSPAYLDY 21 H135 5-51 (3)SYWIG 15 IIYPGDSDTRYSPSFQG 18 GFGASYLDY 22The HC variable region H2 comprises the FR1-CDR1-FR2-CDR2-FR3 derivedfrom SEQ ID NO: 1 where X₁=A, X₂=G, X₃=I, X₄=P, X₅=I, and X₆=F with anadditional mutation in H-CDR2. The HC from library 551 (H14, H17, andH135) comprise FR1-CDR1-FR2-CDR2-FR3 derived from SEQ ID NO: 3 where,X₁=S, X₂=S or G, X₃=I, X₄=Y, X₅=G and X₆=Y or D. Each of the four HCwere comprised of a unique CDR3 (SEQ ID NOS: 19-22).

TABLE 6 Frame-work (SEQ ID SEQ ID SEQ ID SEQ ID VL ID NO:) CDR1 NO: CDR2NO: CDR3 NO: IGKV4-1 B3 (8) KSSQSVLYSSNNKNYLA 23 WASTRES 26 QQYYSTPL 27(B3) L2 B3 (8) KSSQSVLYSSNNKNYLA 23 WASTRES 26 QQSFSFPI 28 L12 B3 (8)KSSQSVLSSSNNENWLA 24 WASTRES 26 QQYYSTPL 27 L111 B3 (8)KSSQSVLASSNNNNFLA 25 WASTRES 26 QQYYSTPL 27

The LC variable regions of the selected Fabs all derive from the B3library and include the curated germline sequence represented in theIMGT database as IGKV4-1 (B3) which was used as a starting sequence forthe library diversification as described herein and referencedpublications. Three of the four light chains had differences in CDR1 andhad identical H-CDR2. A consensus sequence for the four selected LCvariable FR1-CDR1-FR2-CDR2-FR3 derived from SEQ ID NO: 8 wherein X₁ isY, S, or A; X₂ is K, E, or N; X₃ is Y, W or F; and X₄ is always W.

Two unique CDR3 sequences were identified. The L-CDR3 can be representedby a consensus sequence (SEQ ID NO: 29) represented by the formulaQ-Q-(S,Y)-(F,Y)-S-(F,T)-PLT.

Example 4 Affinity Reselection

The four V-region pairings, OSMM5, OSMM6, OSMM9 and OSMM10, described inExample 3 were chosen for light chain reselection in order to improveaffinity. To affinity mature large numbers of antibodies from primaryselections in an efficient and expeditious manner, an “in-line”maturation process described in Shi et al. J Mol Biol 397:385-396, 2010and WO09085462A1 and U.S. Ser. No. 12/546,850 was used. In brief, theV_(H) regions of antigen-specific clones obtained in initial rounds ofselection were combined with libraries of the corresponding V_(L)scaffold, in this case the B3 based V-region (SEQ ID NO: 8).

Three new libraries were created, one using the V_(L) library used inthe primary selections as source of diverse V_(L) chains, and twoadditional libraries designed based on a recent analysis of theantigen-antibody complexes of known structure (Raghuanthan et al., J.Mol. Recognit, 2010). Residues were selected for diversification basedon those residues most likely to be involved in the binding of thetarget protein also called specificity determining residue usage (SDRU).In the Vkappa light chains this has been determined to be three regionsof contacts centered around the hypevariable loops as defined by Chothiaand Lesk which differ slightly depending upon whether the target is aprotein, a peptide, or a small hapten. Table 5 shows the V_(L) librariesused for affinity maturation, where B3 is the same library used duringthe discovery phase, Library 2 “SDRM focused” is based on the SDRUresidues and focused diversity, and NNK is a randomized library. In thetable, “X” means any amino acid plus one stop codon generated by a NNKmix.

Table 7 summarizes the diversity in Libraries 1, 2 and 3. Duringanalysis of the final library, some amino acids were identified thatwere not part of the original design and thus were introduced as aconsequence of the synthesis method. For library 2, synthesized usingdinucleotides, these amino acids were: S (position 30c), T (position30d), EK (position 30f, IW (position 32), TV (position 50), I (position92), D (position 93), and F (position 96).

TABLE 7 Library Position 1 2 3 CDR Sequential Kabat pIX B3 de novoSDRM focused SDRM NNK L1 30 30 L L L 31 30a YSHFA RNDGHSY X 32 30b S S S33 30c S RNDGHWSY X 34 30d N RNDGHSTY X 35 30e N N N 36 30f KTNEEKRNDGHWY X 37 31 N N N 38 32 YFHNWDAS IRNDWY X L2 56 50 WSRDYA YWNKTV XL3 97 91 YSHA SYGH X 98 92 YNDSHIFKG ISYGN X 99 93 SNTDGHR DSTER X 10094 TYLVFAS YSHT X 102 96 WYFLIR FYRWL X

Fab-His protein was prepared from third-round panning outputs andmonoclonal Fab-His ELISAs employed to identify individual hits withhigher binding signal than the corresponding parental Fab. Twoconcentrations of biotinylated human antigen were used in these rankingELISAs: 2 nM and 0.2 nM. The binding signal for each parental clone wasset as 100%. There were 22 hits from the affinity maturation of M6 andM9 showing up to a 9-fold (900%) improved binding when compared to theparental Fab comprising the original heavy and light chain V-regions.Some of these new pairings of heavy and light chains were chosen forfurther evaluation.

The affinity (K_(D)) of inline matured mAbs for human and cynomolgusmonkey OSM compared to that of MAB295 control and parental mAbs M6 andM9 are summarized in Table 8 and the CDR composition of those mAbs shownin Table 9. In some cases, a LCV was paired with both H14 and H17.Certain mAbs that were designated candidate therapeutic leads on thebasis of their biophysical properties and functional potencies arehighlighted in gray.

TABLE 8

TABLE 9 LC-CDR Table for Higher Affinity Mabs PAIRED SEQ ID SEQ IDSEQ ID VH ID VL ID L-CDR1 NO: L-CDR2 NO: L-CDR3 NO: H17 IGKV4-1KSSQSVLYSSNNKNYLA 23 WASTRES 26 QQYYSTPLT 27 (B3) H14 L12KSSQSVLSSSNNENWLA 24 WASTRES 26 QQYYSTPLT 27 H17 L171 KSSQSVLSSGNNGNYLA30 KASTRES 42 QQYYSTPLT 27 H17 L172 KSSQSVLSSGSNHNYLA 31 KASTRES 42QQYYSTPLT 27 H14/7 L173 KSSQSVLSSRGNNNYLA 32 KASTRES 42 QQYYSTPLT 27 H17L174 KSSQSVLGSWGNDNYLA 33 KASTRES 42 QQYYSTPLT 27 H17 L175KSSQSVLYSGGNGNYLA 34 KASTRES 42 QQYSTTPLT 45 H14/7 L176KSSQSVLGSWGNGHYLA 35 KASTRES 42 QQYYSTPLT 27 H17 L177 KSSQSVLSSNGNHNYLA36 KASTRES 42 QQYYSTPLT 27 H17 L178 KSSQSVLSSDGNHNYLA 37 KASTRES 42QQYYSTPLT 27 H17 L180 KSSQSVLGSSSNINFLA 38 SASTRES 43 QQYFSTPIT 46 H14L182 KSSQSVLGSGDNRNYLA 39 NASTRES 44 QQYYSTPLT 27 H14 L186KSSQSVLGSGYNRNYLA 40 KASTRES 42 QQYYSTPLT 27 H14 L184 KSSQSVLGSGYNRNYLA40 WASTRES 26 QQYYSTPLT 27 H17 L192 KSSQSVLGSWHNDNYLA 41 KASTRES 42QQYYSTPLT 27

The LC-CDR3 diversity Q-Q-(S,Y)-(F,Y)-S-(F,T)-PLT (SEQ ID NO: 29) in theselected candidates was reduced, and the consensus sequence for there-selected high affinity LC-CDR3 is represented asQQY-(F,Y)-STP-(L,I)-T (SEQ ID NO: 47).

For the re-selected mAbs, the VH of H14 and H17 share a commonFR1-CDR1-FR2-CDR2-FR3 which is given by SEQ ID NO: 48 and comprising SEQID NO: 14 (CDR1) and SEQ ID NO: 17 (CDR2).

Ability to Reduce Human and Cyno A375-S2 Cell Proliferation

To evaluate the in-line matured antibodies, dose-responses wereconducted starting at 5 μg/ml or 1 μg/ml with 1 to 5 dilutions down to0.0016 or 0.00032 μg/ml, respectively. Neutralization by M71 is shown inFIG. 2B. As the concentration of the antibody is increased, theanti-proliferative effect of both human and cynomolgous monkeyOncostatin M is neutralized. The dose-response curves were calculatedusing data from three separate experiments with human (open symbols) andcynomologous monkey (closed symbols) Oncostatin M. Table 10 summarizesthe IC₅₀ (with 95% confidence intervals) of M55, M64, M69 and M71against human and cynomolgous monkey Oncostatin M.

TABLE 10 Human OSM Cyno OSM Antibody IC₅₀ (ng/ml) 95% C.I. IC₅₀ (ng/ml)95% C.I. M55 54.5 42.5 to 70.0 88.4  67.8 to 115.2 M64 162.4 125.7 to210   321.1 272.6 to 378.1 M69 44.6 32.7 to 60.9 50.81 37.4 to 69.1 M7121.9 16.9 to 28.4 39.67 31.2 to 50.4Human gp130 Competition

Competition experiments were carried out between the anti-OSM mAbschosen for inline maturation plus the selected new binders from thetable above and Human gp130 using Surface Plasmon Resonance (SPR) with aBiacore 3000 optical biosensor (Biacore) as described in Example 1.

The biosensor surface was prepared by coupling each test mAb to thecarboxymethylated dextran surface of a CM-5 chip (Biacore, Cat#BR-1000-14) following the manufacturer's instructions. Approximately4,000-15,000 RU (response units) of each test mAb were immobilized inone the instrument's four flow cells. Competition experiments wereperformed at 25° C. in running buffer (DPBS+0.005% P20+3 mM EDTA). HumanOSM (in-house, OSMN1-1) was diluted to 30 nM in running buffer andinjected at 3 μl/min for 3 minutes over each of the flow cells withimmobilized mAb. Following human OSM capture there was a three-minuteinjection (association phase) of either a competing mAb or humangp130-Fc at 300 nM followed by buffer flow (dissociation phase) for 3minutes. The chip surface was regenerated by two 12-second pulses of 100mM H₃PO₄ (Sigma, Cat#7961) at 50 μl/min. The collected data wereprocessed using BIAevaluation software version 3.2 (Biacore). First, thesensorgrams were aligned upon injection of human OSM. Then the bindinglevels (RU) for human OSM, competing mAb or gp130-Fc were recorded. Arise in binding (RU) when a competing mAb or gp130-Fc was injected overan OSM surface indicates there is no competition with the immobilizedtest mAb and vice versa. Flow-cell (Fc1, etc.) immobilized mAbs appearalong the horizontal sample rows of Table 10.

M2 was previously shown to compete with the commercial antibody MAB295which is known not to compete with gp130 binding to OSM. The resultsdemonstrate of the competition binding assays showed that M54, M55, M64,M69 and M71 compete with human gp130-Fc for OSM antigen (Table 11).

TABLE 11 Human gp130 Competition Matured Candidate Lead mAbs. BindingLevel (RU) Competition M2 M54 M69 M71 M2 M54 M69 M71 Sample (Fc1) (Fc2)(Fc3) (Fc4) (Fc1) (Fc2) (Fc3) (Fc4) Buffer −57 31 35 51 Human 319 17 2029 No Yes Yes Yes gp130-Fc MAB295 −83 2903 2390 1989 Yes No No No M6 98511 16 19 No Yes Yes Yes M54 1765 14 21 33 No Yes Yes Yes M55 1417 14 2135 No Yes Yes Yes M64 1758 12 20 36 No Yes Yes Yes M69 1243 13 17 21 NoYes Yes Yes M71 1194 −1 23 32 No Yes Yes Yes M2 −73 3246 2483 2055 YesNo No No Human 310 19 25 34 No Yes Yes Yes gp130-Fc Buffer −52 35 37 55Binding Level (RU) Competition M2 M55 M64 M2 M55 M64 Sample (Fc1) (Fc2)(Fc3) (Fc1) (Fc2) (Fc3) Buffer −54 50 30 Human 251 25 18 No Yes Yesgp130-Fc MAB295 −80 2950 1948 Yes No No M6 926 36 33 No Yes Yes M54 163419 16 No Yes Yes M55 1316 21 19 No Yes Yes M64 1665 20 22 No Yes Yes M691148 17 11 No Yes Yes M71 1127 22 17 No Yes Yes M2 −74 3474 2203 Yes NoNo Human 248 26 20 No Yes Yes gp130-Fc Buffer −47 60 38Ability to Block pSTAT3 in A375 Cells

The calculated EC50 values for pSTAT3 inhibition performed in A375 cellsin the presence or absence of 5 ng/ml hOSM for M6 and M9 and in-linematured variants of these Mabs, are shown in Table 12.

TABLE 12 pSTAT3 inhibition EC50 95% Confidence Curve mAb ID (ng/ml)Intervals Fit R² M65 29.5 24.35 to 35.82 0.9921 M67 37.7 30.93 to 45.920.9905 M45 50.0 42.45 to 58.91 0.9931 M83 71.2 59.59 to 85.05 0.9908MAB295 72.1 46.91 to 110.7 0.9848 M54 78.5 66.11 to 93.23 0.9930 M6388.2 58.55 to 132.9 0.9819 M64 97.1 77.01 to 122.4 0.9902 M68 126.9107.2 to 150.1 0.9924 M69 141.7 115.1 to 174.5 0.9914 M66 152.7 111.1 to209.8 0.9833 M71 159.6 128.3 to 198.5 0.9885 M55 181.6 153.0 to 215.60.9869 M42 191.3 93.67 to 390.7 0.9607 M62 233.5 206.4 to 264.1 0.9947M53 236.6 196.3 to 285.2 0.9859 M6 650.3 241.7 to 1750 0.9540 M9 1218.0352.8 to 4208 0.9614 M85 — 0.4454 IL13 — 0.3438

Example 5 Biologic Activity Macrophage-Chondrocyte Co-Culture Assay

M71 was evaluated in a macrophage-chondrocyte co-culture system.Differentiated macrophages are known to produce Oncostatin M (Hasegawaet al. Rheumatology 38:612-617, 1999). OSM can decrease the synthesis ofthe highly-sulfated proteoglycan aggrecan (GAG) that makes up asignificant portion of the cartilage matrix.

The anti-human Oncostatin M antibodies were discovered using recombinantHEK-generated His-Avi tagged human and cynomolgous monkey Oncostatin Mand the activity of these antibodies was evaluated using these moleculesas well as bacterial-derived recombinant human Oncostatin M from R&DSystems (295-OM). None of these are identical to native endogenous humanOncostatin M, either because of the his-avi tag (HEK-generated Osm) orthe lack of glycosylation (bacterial recombinant Osm). Macrophages areknown to secrete Oncostatin M (Grove et al., J Lipid Res 32:1889-97,1991) and oncostatin M decreases proteoglycan synthesis in humanchondrocytes (Sanchez et al. OA and Cart. 12:810-10, 2004). Thus, amacrophage-chondrocyte co-culture system was used to determine theability of the anti-human Oncostatin M antibodies to neutralizeendogenous, or native, human Oncostatin M. Briefly, single alginatebeads containing 40,000 normal human articular chondrocytes werecultured for 72 hours in the presence of differentiated humanmacrophages. These experiments were conducted in the presence of adose-range of anti-Oncostatin M antibody. At the end of the experiment,proteoglycan synthesis was measured by the incorporation of radioactive³⁵SO₄.

CD14+ peripheral blood monocytes were obtained from AllCells. Themonocytes were cultured on 48-well plates at 2.5×105 cells/well in 0.5mls of macrophage medium (RPMI+Glutamine with 10% heat inactivated FBS,1% NEAA and 1% Pen-Strep). The cells were treated with 100 ng/ml ofmacrophage-colony stimulating factor (M-CSF). After 48 hours the mediawas replaced to remove non-adherent cells. On day 6, the M-CSFcontaining macrophage medium was replaced with macrophage medium withoutM-CSF. On Day 8, the macrophage medium was replaced with chondrocytemedium (50% Ham's F-12/50% DMEM with 10% fetal calf serum) and a singlealginate bead chondrocyte culture (Articular Engineering #CDD-H-2200)was added to each well. The aliquots of conditioned macrophage mediumwere reserved and stored at −80° C. for analysis of Oncostatin M levelsusing the R&D Systems Human Oncostatin M DuoSet (DY295).

The alginate bead-macrophage co-cultures were maintained in the presenceof either 20 μg/ml of anti-human Oncostatin M antibodies (M64, M71, M55,M69) or in the presence of a dose-range (5 μg/ml to 0.00076 μg/ml; 1 to3 dilutions) of antibody (M71 and M55). In addition, human IgG1 isotypecontrol (CNTO6234) was included on the plate at the highestconcentration of anti-oncostatin M antibody tested. Other controls werechondrocytes only (no co-culture) and chondrocytes in the presence of 2ng/ml human Oncostatin M. In addition, wells containing macrophages onlywere maintained in order to measure Oncostatin M production. After 72hours of co-culture, 10 μCi/ml radioactive 35SO4 (Perkin-ElmerNEX041H002MC) was added to each well for an additional 20 hours.

Incorporation of ³⁵SO₄ was measured using the CPC precipitationtechnique (MP BIomedicals (#190177). After 20 hours of incubation with³⁵SO₄, the labeled media was removed and each bead was washed twice withDPBS with Ca and Mg. After the second wash, 200 μl of citrate buffer(150 mM NaCl, 55 mM NaCitrate, ph 6.8) was added to each bead. Theplates were incubated for 10 to 15 min at 37° C. until the beads weredissolved. A 100 μl aliquot from each well was transferred to aMillipore Multiscreen 96-well filter plate prewetted with 1% CPC andthen 10 μl of 10% CPC was added to each well for 5 minutes. Vacuum wasthen applied to the plate until the filters were dry. Each well was thenwashed 2 times with 200 μl of 1% CPC and vacuum was applied each timeuntil the filters were dry. The plastic bottom was then removed from theplate and replaced with a sealer (Perkin Elmer #6005185). Scintillationfluid (50 μl, Perkin Elmer #6013621) was added to each well and a platesealer was applied to the top of the plate. The plate was then countedon a Top Count reader.

At 20 μg/ml, M64, M71, M55, and M69 increased proteoglycan synthesisabove the level observed in the absence of antibody while the isotypecontrol had no effect (FIG. 3). In a separate experiment, M71dose-dependently increased proteoglycan synthesis to the level exhibitedby chondrocytes alone (defined as 100% neutralization), with an EC50 of30 ng/ml and the isotype control had no effect. These data demonstratethat macrophage-derived Osm decreases proteoglycan synthesis in theco-cultured chondrocytes and that the anti-human Oncostatin M antibodiesneutralize native Oncostatin M.

Human Lung Fibroblast Phospho-STAT3 Assay

OSM induces proliferation and collagen production in normal human lungfibroblasts (Scaffidi et al., Br. J. Pharmacol 136: 793-801, 2002).Over-production of collagen by fibroblasts is a key feature of a numberof pathological conditions (Lim et al. Oncogene 23(39): 5416-25, 2006;Huang et al. J Cell Biochem 81(1): 102-13, 2001). Oncostatin M receptorsignaling activates the JAK-STAT pathway and phosphorylation of STAT3 isan early event in the signaling pathway (Auguste et al. (1997) Signalingof Type II Oncostatin M Receptor. J Biol Chem 272:15760-15764). Theability of Oncostatin M to generate pSTAT3 was determined in normalhuman lung fibroblasts (NHLF) using the R&D Systems human/mouse pSTAT3Duoset (DYC4607-5). This assay was then used to determine the ability ofM55 and M71 to neutralize Oncostatin M signaling.

These experiments were carried out using NHLF from Lonza (CC-2512) grownin Lonza proprietary FGM-2 (CC-3132) media. Briefly, the cells wereplated at 25,000 cells/well in FGM-2 and cultured for 24 hours. Thecells were then treated with Oncostatin M or antibody plus Oncostatin Mfor 10 minutes. In order to avoid temperature dependent effects duringthis 10 minute incubation, all of the solutions used to prepare thetreatments were pre-warmed and maintained at 37° C. After the 10 minutetreatment, media was aspirated away and replaced with complete lysisbuffer. The lysis buffer (pH=7.2) consisted of 1% NP-40, 1% sodiumdeoxycholate, 0.1% SDS, 0.15M NaCl and 0.01M sodium phosphate and wasstored at 4° C. At the time of use, complete lysis buffer was preparedby adding 1 tablet of Protease Inhibitor Cocktail (Roche, 11836153001)and 110 μL HALT phosphatase inhibitor (Thermo Scientific 78420) to 11mls of lysis buffer. After the 10 minute lysis step, the resultinglysate was ready for pSTAT3 detection.

To determine the Oncostatin M dose-response, NHLF cells were treatedwith a dose-range of OSM (100 ng/ml to 0.024 ng/ml w/1 to 4 dilutions,triplicate wells). A dilution plate was prepared in pre-warmed PBS+1%BSA in which the concentration of Osm in each well was 10× higher thanthe final concentration needed for treatment, with media-only wellsincluded as untreated controls. The media was completely removed fromthe culture plate and replaced with 180 μl of pre-warmed FGM-2. A timerwas started for 10 minutes and then 20 μl was transferred from each wellin the dilution plate to the corresponding well in the culture plate.After 10 minutes, the treatment solutions in the cell plate werecompletely removed by aspiration and replaced with 100 μl of completelysis buffer. To minimize differences in incubation time, the lysisbuffer was added to wells in the same order as the treatments. The assayplate was then placed on a shaker for 10 minutes. After shaking, thelysates were either frozen at −80° C. for later testing or transferreddirectly to an ELISA plate coated with anti-pSTAT3. The ELISA (R&DSystems human/mouse pSTAT3 Duoset) was carried out according to themanufacturer's instructions with the exceptions that 1) only 90 μl oflysate or standard was added to each well and 2) SuperSignal Pico(ThermoScientific 37069) was used as the HRP substrate. The ELISA platewas read for luminescence on a Victor3 plate reader.

To evaluate the ability of the M55 and M71 to neutralize OSM signalingin NHLFs, the cells were treated with a dose-range of anti-OSM antibody(triplicate wells, 500 ng/ml to 0.005 ng/ml with 1-10 dilutions or 50ng/ml to 1.563 ng/ml with 1 to 2 dilutions) in the presence of 2 ng/mlhuman Osm. A dilution plate was prepared in PBS for the antibodydose-response treatment at 20× the final concentration, with wellsincluded for the isotype control (at the highest concentration of theanti-OSM antibody) as well as with no antibody for both the untreatedcontrol and the cells treated only with OSM. Human oncostatin M wasprepared separately at 40 ng/ml (20× the final concentration) in FGM-2.The 20× antibody and OSM solutions were mixed in equal volumes in thedilution plate to generate 10× treatment solutions and the plate wasincubated for 1 hour at 37° C. to allow OSM to bind to the antibody.After 1 hour, the media was removed from the culture plate and replacedwith 180 μl of pre-warmed FGM-2. A timer was started for 10 minutes and20 μl was transferred from each well in the dilution plate to thecorresponding well in the culture plate. After 10 minutes, the solutionsin the cell plate were completely removed by aspiration and replacedwith 100 μl of complete lysis buffer. To minimize differences inincubation time, the lysis buffer was added to wells in the same orderas the treatments. The assay plate was then placed on a shaker for 10minutes. After shaking, the lysates were either frozen at −80° C. forlater testing or transferred directly to an ELISA plate coated withanti-pSTAT3. The ELISA (R&D Systems human/mouse pSTAT3 Duoset) wascarried out according to the manufacturer's instructions with theexceptions that 1) only 90 μl of lysate or standard was added to eachwell and 2) SuperSignal Pico (ThermoScientific 37069) was used as theHRP substrate. The ELISA plate was read for luminescence on a Victor3plate reader.

Human Oncostatin M increased pSTAT3 in NHLF cells with an EC50 ofapproximately 1 ng/ml. An example of an Oncostatin M dose response isprovided in FIG. 4A. For this example, the EC50 was 0.90 ng/ml with a95% confidence interval from 0.70 to 1.17 ng/ml. The ability of theanti-oncostatin M antibodies to neutralize was determined in thepresence of 2 ng/ml of Oncostatin M and all of the data were normalizedto the luminescence in the presence of 2 ng/ml OSM with no antibodypresent. FIG. 4B shows the dose-dependent neutralization of OSM-inducedSTAT3 phosphorylation by M71. As the concentration of M71 is increased,the extent of STAT3 phosphorylation decreases. The dose-response curvewas calculated from data from six separate experiments and thecalculated IC50 for M71 was 8.9 ng/ml with a 95% confidence intervalfrom 6.9 to 11.6 ng/ml.

Example 6 In Vivo Activity

M71 was evaluated for its ability to block the production of cytokinesin vivo after systemic administration human Oncostatin M.Intraperitoneal injection of human Oncostatin M increases levels ofcertain serum cytokines, presumably through and interaction with themurine leukemia inhibitory factor receptor.

Systemic (i.p.) administration of human oncostastin M to mice wasdeveloped as a model to evaluate the ability of the anti-oncostatin Mmonoclonal antibodies to neutralize in an in vivo setting. Mice wereinjected i.p. with 10 μg of human Oncostatin M in 200 μl of PBS or PBSvehicle control. After 1 hour the mice were anesthetized with CO2 andblood was collected by terminal cardiac puncture. The individual bloodsamples were allowed to clot on ice for 20 min and then spun at 3500 rpmfor 10-15 minutes. Serum samples were kept frozen until analyzed, as perthe manufacturer's instructions, with the Milliplex Murine MAPCytokine/Chemokine Multiplex (32) Panel. Analysis of the samplesdemonstrated that, compared to vehicle control, human Oncostatin Msignificantly increased serum levels of murine KC, IP-10, MCP-1, IL-6and eotaxin, with no effect on the other cytokines in the panel. Thesedata demonstrate that injection of human Oncostatin M induces cytokinerelease, presumably through an interaction with the murine leukemiainhibitory factor receptor (Richards et al. J. Immunol. 159:2431-37,1997; Lindberg et al., Mol Cell Biol 18:3357-3367, 1988) that can beused to test the ability of the anti-Oncostatin M antibodies toneutralize in vivo.

M71 and M55 were evaluated in the mouse systemic administration model.Briefly, mice were dosed subcutaneously with M71 or M55 (human IgG1anti-human Osm at 20, 2.0 or 0.2 mg/kg), CNTO6234 (huIgG1 isotypecontrol, 20 mg/kg) or PBS in a volume of 10 μl/g. After 24 hours, eachmouse was then injected i.p. with either 10 μg of in-house generated CHOcell-derived recombinant human oncostatin M in PBS (Sigma D8357) w/0.1%mouse serum albumin (Sigma A3559) or with PBS-MSA vehicle control alone(200 μL total volume). After 1 hour the mice were anesthetized with CO2and blood was collected by terminal cardiac puncture. The individualblood samples were allowed to clot on ice for 20 min and then spun at3500 rpm for 10-15 minutes. Serum samples were kept frozen untilanalyzed using the Milliplex Murine MAP Cytokine/Chemokine Multiplex(32) Panel. Serum samples were analyzed as per the manufacturer'sinstructions.

Human oncostatin M induced significant (unpaired Student's t-test)increases in the serum levels of five of the cytokines detected by thekit: eotaxin, IL-6, IP-10, KC and MCP-1. Pre-dosing with the isotypecontrol CNTO6234 at 20 mg/kg had no effect on oncostatin M-inducedcytokine release. However, pre-dosing with M71 significantly reducedserum levels of IP-10, MCP-1, IL-6 and eotaxin at 2.0 and 20 mg/kg andKC at 20 mg/kg. There was no effect on any of the cytokines at 0.2 mg/kgof M71. The effect of M71 and the isotype control on IP-10 and MCP-1 areshown in FIGS. 5A and B, respectively. Less robust neutralization wasobserved with M55 as neutralization at both 20 and 2.0 mg/kg was seenonly with IP-10. Serum levels of IL-6, eotaxin and MCP-1 were reduced at20 mg/kg M55 but no reduction in KC levels were seen at any dose of M55.These data demonstrate the ability of the anti-Oncostatin M antibodiesto neutralize the biological effects of exogenous human oncostatin M inthe murine systemic administration model.

IP-10, interferon gamma-induced protein 10 kDa or small-induciblecytokine B10, is a protein that in humans is encoded by the CXCL10 gene(C-X-C motif chemokine 10 (CXCL10). CXCL10 has been attributed toseveral roles, such as chemoattraction for monocytes/macrophages, Tcells, NK cells, and dendritic cells, and promotion of T cell adhesionto endothelial cells. KC, now known as chemokine (C-X-C motif) ligand 1(CXCL1), is a small cytokine belonging to the CXC chemokine family thatwas previously called GRO1 oncogene, GROα, Neutrophil-activating protein3 (NAP-3) and melanoma growth stimulating activity, alpha (MSGA-α). Inhumans, this protein is encoded by the CXCL1 gene. CXCL1 is expressed bymacrophages, neutrophils and epithelial cells, and has neutrophilchemoattractant activity.

Example 7 Co-Crystallography

A Fab fragment comprising the V-regions H17 (SEQ ID NO: 51) and L180(SEQ ID NO: 55) was crystallized with human OSM (SEQ ID NO: 10) residues26-212.

OSM shares its four-helix bundle three-dimensional structure with othermembers of the gp130 cytokine family. The four-helix bundle architectureis characterized by four α helical segments designated A (residues10-37), B (residues 67-90), C (residues 105-131) and D (residues159-185) linked by relatively unstructured loops. OSM interacts withgp130 via an epitope termed Site II which includes amino acid residues(Q16, Q20, G120, N123, N124) located in helices A and C (Deller et al.Structure 8(8): 863-874, 2000; Liu et al. Int. J. Mol. Med. 23: 161-172,2009). Site III, the epitope responsible for OSM interaction with OSMRβand LIFRα, is believed to be largely defined by residues located inhelix D (Deller et al. Structure 8(8): 863-874, 2000).

Crystallization

Crystallization of the complex was performed by the sitting-dropvapor-diffusion method at 20° C. using the Oryx4 protein crystallizationrobot (Douglas Instruments), dispensing equal volumes of 0.2 μL proteincomplex (10.95 mg/mL) and 0.2 μL reservoir solution. Multiplecrystallization screens were performed. The majority of dropletsremained clear, reflecting the high solubility of the complex. Crystalswere obtained from 0.1 M MES pH 6.5, 2.4 M ammonium sulfate and 0.1 MTris pH 8.5, 3.5 M sodium formate.

Result of Crystal Structure Solution

The OSM residues in contact with H14/L180 Fab constitute the bindingepitope. The antibody residues in contact with OSM constitute thebinding paratope. All six CDRs of the two variable domains are involvedin OSM binding. Residues in contact are given in Table 13 and shown inFIG. 5. The long CDR-L1, together with the CDRs of the heavy chainvariable region H1, form a valley-like antigen binding site with a smallridge at the bottom (FIG. 4C, left panel). Upon binding, the two sidesof the valley embrace the OSM four-helix bundle along helices A and Cwith the bottom ridge binding in between the two helices (FIG. 4C, rightpanel). The antibody and antigen binding interface buries 2,514 Å2 ofsolvent accessible surface (1225 Å2 for Ab and 1298 Å2 for Ag). Althoughthere are a number of charges residues at the interface, there is nocharge-charge pairing, suggesting vdw and H-bonding play the mostimportant roles in antibody and antigen interactions.

TABLE 13 OSM VH (H17) VL (L180) Q16 Y32, R98 K19 Y105 Y55, E61 Q20 S31,P100, V101 D22 I36 L23 P100, V101, A104, Y105 Y97 Q25 S34 D26 G31, S32,S33 T27 S32, S33 S28 S32, F38, F98 R29 F38, Y97, F98 D32 S32 I105 R59E106 T100 E109 Y57, R59 K110 F98, S99 Q112 Y57 M113 Y57, R59, S102 T100P116 W33, Y52 N117 W33, V101, S102 L119 Y52, D55 G120 T30, Y52 * Thedistance cutoffs are 3.3 Å for H bonds (highlighted in bold) and 3.9 Åfor vdw contacts.The H17/L180 Fab thus contacts OSM at residues previously shown to becontacted by 130; Q20 and G120, and along the A and C helices.

Example 8 Pharmacokinetics

OSM is a soluble target associated with the inflammatory processes asopposed to a cell-surface displayed target on a cell. The nature of acomplete IgG comprising the binding regions as discovered herein andadditionally having an Fc domain can thus be tailored to the purpose andtherapeutic specification related to its use using methods ofengineering the Fc with mutations conferring altered FcR binding.

In the present composition, a sustained activity and persistence in thecirculation is a beneficial specification of a therapeutic monoclonalIgG. Therefore, an Fc-domain having enhanced affinity for the neonatalreceptor (FcRn).

Using mutations previously described, M428L (MedImmune U.S. Pat. No.7,670,600), in combination with N434S (U.S. Pat. No. 7,371,826,WO2006/053301), a mutated and a wild-type antibody were constructedusing standard recombinant techniques. These two Mabs, M71 and M71 L/Swere compared in standard activity assays and compared for persistencein the circulation of a non-human primate.

Assays

M71 and M71 L/S were compared side-by-side in the A375-S2 proliferationassay. Dose-responses were evaluated from a starting concentration of 1μg/ml, with 1 to 5 dilutions to 0.00032 μg/ml. At a concentration of 1μg/ml the isotype controls for these antibodies, CNTO3930 and CNTO8852,respectively, had no effect on the ability of 2 ng/ml of humanOncostatin M to inhibit proliferation. Both M71 and M71 L/S completelyneutralized the effect of Oncostatin M at 1 μg/ml, with no measurabledifference in IC50.

M71 and M71 L/S were compared in the mouse systemic administrationmodel. Briefly, mice were dosed subcutaneously with M71 and M71 L/S (20,or 5.0 mg/kg), CNTO3930 (huIgG1 isotype control, 20 mg/kg), CNTO8852(isotype control for Fc mutated version, 20 mg/kg) or PBS in a volume of10 μl/g. After 24 hours, each mouse was injected i.p. with either 10 μgof in-house generated CHO cell-derived recombinant human oncostatin M inPBS (Sigma D8357) w/0.1% mouse serum albumin (Sigma A3559) or withPBS-MSA vehicle control alone (200 μL total volume). After 1 hour themice were anesthetized with CO2 and blood was collected by terminalcardiac puncture. The individual blood samples were allowed to clot onice for 20 min and then spun at 3500 rpm for 10-15 minutes. Serumsamples were kept frozen until analyzed using a custom Millipore murinemultiplex consisting of beads specific for IL-6, MCP-1, eotaxin, KC andIP-10. Neither isotype control had any effect on cytokine releaseinduced by human oncostatin M. However, both M71 and M71 L/S neutralizedoncostatin M-induced cytokine release, with no apparent differences inpotency or efficacy.

Pharmacokinetic Analysis

The serum half-life of M71 and M71 L/S were compared in a non-terminalcynomolgous monkey pharmacokinetics study. The study included a total of12 cynomolgous monkeys and evaluated subcutaneous (s.c., n=3) andintravenous (i.v., n=3) administration for each antibody. The antibodieswere dosed at 3 mg/kg and the study was carried out over 60 days. Bloodsamples were taken at 1 (IV groups only) and 6 hours and on days 1, 2,4, 6, 8, 12, 16, 30, 37, 45 and 60. Serum from the samples was frozen at−80° C. until testing. Antibody levels in the serum were determinedusing an ELISA optimized in cynomologous monkey serum for the MesoScaleDiscovery platform. The biotinylated capture antibody was ananti-idotype antibody raised against M71 (mouse anti-M71). The detectionantibody was ruthenium-labeled anti-human IgG and the readout wasMesoScale Discovery chemiluminescence.

The results of the study are shown in FIGS. 6A and B. FIG. 6A shows theplots from the i.v. dosing where the serum half-life of M71 was15.21+/−3.0 days and the half-life of M71 L/S was 29.4+/−2.3 days.Similar results were obtained from s.c. dosing (FIG. 6B) with a serumhalf-life of 15.4+/−4 days for M71 and 32.0+/−5.9 days for M71 L/S.

The results showed that M71 L/S t ½ increased about two-fold as comparedto M71.

1. An isolated antibody that specifically binds to human OSM protein andmodulates the interaction between human OSM protein and human gp130protein, comprising a heavy chain complementarity determining region 3(H-CDR3) selected from the amino acid sequence of SEQ ID NOS: 20 and 21.2. The antibody of claim 1, further comprising a heavy chain framework 1sequence, H-CDR1 sequence, framework 2 sequence, H-CDR2 sequence, andframework 3 sequence having the amino acid sequence of SEQ ID NO:
 48. 3.The antibody of claim 2, wherein the heavy chain residue S31 contactsthe OSM protein having the amino acid sequence of SEQ ID NO: 11 atposition Q20 and heavy chain residues T30 and Y52 contact the OSMprotein having the amino acid sequence of SEQ ID NO: 11 at positionG120.
 4. The antibody of claim 2, wherein residues of the human variablelight chain framework are derived from a human Vkappa germline gene. 5.The antibody of claim 4, wherein the Vkappa germline gene is an IGKV4germline gene sequence.
 6. The antibody of claim 2, further comprising alight chain variable region comprising SEQ ID NO: 8, wherein X₁ is Y, S,or A; X₂ is K, E, or N; X₃ is Y, W or F; and X₄ is W.
 7. The antibody ofclaim 6, wherein the L-CDR3 sequence comprises an amino acid sequence ofSEQ ID NO:
 29. 8. The antibody of claim 7, wherein the L-CDR3 sequencecomprises an amino acid sequence selected from the group consisting ofSEQ ID NOS: 27, 28, 45, and
 46. 9. The antibody of claim 6, wherein theL-CDR1 sequence comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 23-25 and 30-41.
 10. The antibody of claim 9,wherein the L-CDR2 sequence comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 26 and 42-44.
 11. An isolatedantibody that specifically binds to human OSM protein and modulates theinteraction between human OSM and human gp130 protein, comprising alight chain variable region amino acid sequence selected from the groupconsisting of SEQ ID NOS: 49-53 and/or a heavy chain variable regionamino acid sequence selected from the group consisting of SEQ ID NOS: 54and
 55. 12. The isolated antibody of claim 11, wherein the antibodycomprises a light chain variable region amino acid sequence of SEQ IDNO: 53 and a heavy chain variable region amino acid sequence SEQ ID NO:54.
 13. The isolated antibody of claim 11, wherein an antibody paratopecontacts epitope residues of human OSM protein having the amino acidsequence of SEQ ID NO: 11 at positions Q16, Q20, and G120.
 14. Anisolated antibody comprising a light chain variable region amino acidsequence of SEQ ID NO: 51 and a heavy chain variable region amino acidsequence SEQ ID NO:
 55. 15. The isolated antibody of claim 14, furthercomprising a human heavy chain constant region selected from the groupconsisting of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM. 16.The isolated antibody of claim 15, wherein the constant region comprisesa human IgG isotype.
 17. The isolated antibody of claim 16, wherein theisotype is IgG1.
 18. The isolated antibody of claim 17, wherein theconstant region is mutated to render the antibody non-lytic.
 19. Theisolated antibody of claim 16, wherein the constant region is mutated toenhance the affinity of the antibody to the neonatal receptor (FcRn) ascompared to an antibody with the wild-type IgG1 constant domainsequence.
 20. The isolated antibody of claim 19, wherein the constantregion is mutated at positions 428 and 434, wherein the numbering isaccording to the Kabat EU numbering.
 21. The isolated antibody of claim20, wherein the mutations are M428L and N434S, wherein the numbering isaccording to the Kabat EU numbering.
 22. An antigen binding fragment ofthe antibody of claim
 14. 23. The antigen binding fragment of claim 22,wherein said fragment is selected from the group consisting of a Fab,Fab′, Fd, F(ab)₂, and ScFv.
 24. A pharmaceutical composition comprisingthe isolated antibody of claim 14 and a pharmaceutically acceptableexcipient or carrier.
 25. A method of treating a human patient afflictedwith a disease or disorder responsive to modulation of the interactionbetween human OSM protein and human gp130 protein, said methodcomprising the step of administering to said patient a therapeuticallyeffective amount of the composition of claim
 24. 26. The method of claim25, wherein the disease or disorder is an arthropathy selected form thegroup consisting of osteoarthritis, rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, neuropathic arthropathy, reactivearthritis, and rotator cuff tear arthropathy.
 27. A method of treating ahuman patient afflicted with a disease or disorder characterized by therelease of pro-inflammatory cytokines and chemokines by macrophages andmonocytes, comprising the step of administering to said patient atherapeutically effective amount of the composition of claim
 24. 28. Amethod according to claim 25, wherein the disease or disorder isselected from the group consisting of rheumatoid arthritis, juvenileonset arthritis, psoriatic arthritis, ankylosing spondylitis, psoriasis,chronic plaque disease, lupus erythematosus, inflammatory lung disease,idiopathic pulmonary fibrosis, sepsis, pre-eclampsia, COPD, asthma, andmultiple sclerosis.
 29. A method of treating a human patient accordingto claim 25, wherein the patient is afflicted with a fibrotic diseaseselected from the group consisting of atherosclerosis, diabeticnephropathy, pulmonary fibrosis, idiopathic pulmonary fibrosis, systemicsclerosis, and cirrhosis.
 30. An isolated polynucleotide encoding theheavy chain and/or light chain of the isolated antibody of claim
 14. 31.A stably transformed or transfected recombinant host cell comprising theisolated polynucleotide of claim
 30. 32. The stably transformed ortransfected recombinant host cell of claim 31, comprising a vectorcomprising a polynucleotide encoding the light chain variable amino acidsequence of SEQ ID NO: 53 and a second polynucleotide encoding the heavychain variable amino acid sequence of SEQ ID NO:
 54. 33. The stablytransformed or transfected recombinant host cell of claim 31, comprisinga vector comprising a polynucleotide encoding the light chain variableamino acid sequence of SEQ ID NO: 51 and a second polynucleotideencoding the heavy chain variable amino acid sequence of SEQ ID NO: 55.34. The host cell of claim 33, wherein said cell is mammalian.
 35. Thehost cell of claim 34, wherein said cell is CHO.
 36. A process for themanufacture of an antibody, comprising the steps of culturing a hostcell of claim 34 and recovering the antibody from the cell.
 37. A kitcomprising a sterile preparation of the isolated antibody of claim 14and instructions for the administration of the antibody to a subject inneed thereof.
 38. An isolated antibody that specifically binds to humanOSM protein, comprising: a. a light chain complementarity determiningregion 1 (L-CDR1) amino acid sequence selected from the group consistingof SEQ ID NOS: 23-25, and 30-41; b. a light chain complementaritydetermining region 2 (L-CDR2) amino acid sequence selected from thegroup consisting of SEQ ID NOS: 26, 42-44; and c. a light chaincomplementarity determining region 3 (L-CDR3) amino acid sequenceselected from the group consisting of SEQ ID NOS: 10, 27-29, 45,
 47. 39.An isolated antibody that specifically binds to OSM, comprising: a. aheavy chain complementarity determining region 1 (H-CDR1) amino acidsequence selected from the group consisting of SEQ ID NOS: 14 and 15; b.a heavy chain complementarity determining region 2 (H-CDR2) amino acidsequence selected from the group consisting of SEQ ID NOS: 16-18; and c.a heavy chain complementarity determining region 3 (H-CDR3) amino acidsequence selected from the group consisting of SEQ ID NOS: 19-22.
 40. Anisolated antibody that specifically binds to OSM, comprising the H-CDR1,H-CDR2 and HCDR3 of claim 39 and: a. a light chain complementaritydetermining region 1 (L-CDR1) amino acid sequence selected from thegroup consisting of SEQ ID NOS: 23-25, and 30-41; b. a light chaincomplementarity determining region 2 (L-CDR2) amino acid sequenceselected from the group consisting of SEQ ID NOS: 26, 42-44; and c. alight chain complementarity determining region 3 (L-CDR3) amino acidsequence selected from the group consisting of SEQ ID NOS: 10, 27-29,45,
 47. 41. An isolated antibody that specifically binds to OSM,comprising: an H-CDR1 having an amino acid sequence of SEQ ID NO: 14; anH-CDR2 having an amino acid sequence of SEQ ID NO: 17; an H-CDR3 havingan amino acid sequence of SEQ ID NO: 21; an L-CDR1 having an amino acidsequence of SEQ ID NO: 38; an L-CDR2 having an amino acid sequence ofSEQ ID NO: 43; and an L-CDR3 having an amino acid sequence of SEQ ID NO:46.
 42. The isolated antibody of claim 41, further comprising a humanheavy chain constant region selected from the group consisting of IgA1,IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM.
 43. The isolatedantibody of claim 42, wherein the constant region comprises a human IgGisotype.
 44. The isolated antibody of claim 43, wherein the isotype isIgG1.
 45. The isolated antibody of claim 44, wherein the constant regionis mutated to render the antibody non-lytic.
 46. The isolated antibodyof claim 43, wherein the constant region is mutated to enhance theaffinity of the antibody to the neonatal receptor (FcRn) as compared toan antibody with the wild-type IgG1 constant domain sequence.
 47. Theisolated antibody of claim 46, wherein the constant region is mutated atpositions 428 and 434, wherein the numbering is according to the KabatEU numbering.
 48. The isolated antibody of claim 47, wherein themutations are M428L and N434S, wherein the numbering is according to theKabat EU numbering.
 49. An antigen binding fragment of the antibody ofclaim
 41. 50. The antigen binding fragment of claim 49, wherein saidfragment is selected from the group consisting of a Fab, Fab′, Fd,F(ab)₂, and ScFv.
 51. A pharmaceutical composition comprising theisolated antibody of claim 41 and a pharmaceutically acceptableexcipient or carrier.
 52. An isolated polynucleotide encoding the heavychain and/or light chain of the isolated antibody of claim
 41. 53. Astably transformed or transfected recombinant host cell comprising theisolated polynucleotide of claim
 52. 54. The stably transformed ortransfected recombinant host cell of claim 53, comprising a vectorcomprising a polynucleotide encoding an L-CDR1 having an amino acidsequence of SEQ ID NO: 38, an L-CDR2 having an amino acid sequence ofSEQ ID NO: 43, and an L-CDR3 having an amino acid sequence of SEQ ID NO:46; and a second polynucleotide encoding an H-CDR1 having an amino acidsequence of SEQ ID NO: 14, an H-CDR2 having an amino acid sequence ofSEQ ID NO: 17, and an H-CDR3 having an amino acid sequence of SEQ ID NO:21
 55. The host cell of claim 54, wherein said cell is mammalian. 56.The host cell of claim 55, wherein said cell is CHO.
 57. A process forthe manufacture of an antibody, comprising the steps of culturing a hostcell of claim 55 and recovering the antibody from the cell.
 58. Anyinvention described herein.